Background Sustained minimal residual disease (MRD) negativity is associated with improved myeloma (MM) outcomes. The gut microbiome can modulate host immunity through production of short-chain fatty acids, especially butyrate. Stool butyrate-producers are enriched in MRD negative MM (Eubacterium, Faecalibacterium; Pianko et al. 2019) and are associated with post-transplant survival (Eubacterium; Peled et al. 2017). However, microbial features associated with sustained MRD negativity and diet which may impact microbiome and in turn MM outcomes have not been explored. Herein we evaluate the association of diet and microbiome with sustained MRD negativity in MM in the context of lenalidomide maintenance. Methods MM patients eligible for lenalidomide maintenance during first-line therapy were enrolled (NCT02538198). MRD status was evaluated at baseline and annually with BM flow-cytometry (sensitivity >10-5). Sustained MRD negativity was defined as MRD negative for 1 year from enrollment. The relative abundance of butyrate producers and microbiome α-diversity were calculated from fecal 16S microbiome profiles at baseline, 3 months (m) and annually, along with quantification of fecal butyrate levels at 3m. Dietary patterns were collected using the Block Food Frequency Questionnaire (FFQ), for habitual diet and summarized via the Healthy Eating Index 2015 (HEI2015) scores in which higher scores imply healthier diets. The association between dietary and microbiome data were evaluated by Spearman’s rank correlation coefficient. Logistic regression was used to evaluate association between MRD status at 1 year (Y) and microbiome and dietary measurements. Statistical significance was defined using a two-sided significance level at 0.05. Results We had 49 patients with stool samples at 3m, 30 patients at Y1, 32 with stool butyrate levels at 3m, 59 with FFQ data, and the overlap between FFQ and stool samples was 34. The abundance of butyrate producers and/or butyrate levels were significantly associated with total HEI2015 and its components – total vegetables, dark green vegetables, legumes, whole grains, total protein, seafood and plant protein, fatty acids, refined grains, added sugars. Some aspects of healthier HEI score components (especially seafood and plant proteins) were associated with higher stool butyrate producers and butyrate levels at 3m, and sustained MRD negativity at Y1. Additionally, increased diversity, butyrate levels and butyrate producers at 3m was associated with increased MRD negativity at baseline and with sustained MRD negativity. Conclusion In MM patients on lenalidomide maintenance, healthier diets (especially seafood and plant proteins) were associated with increased butyrate at 3m and in turn with higher MRD negativity. This is the first study to demonstrate an association between diet, microbiome, and sustained MRD negativity and provides a rationale for evaluating a targeted dietary intervention prospectively. Sustained minimal residual disease (MRD) negativity is associated with improved myeloma (MM) outcomes. The gut microbiome can modulate host immunity through production of short-chain fatty acids, especially butyrate. Stool butyrate-producers are enriched in MRD negative MM (Eubacterium, Faecalibacterium; Pianko et al. 2019) and are associated with post-transplant survival (Eubacterium; Peled et al. 2017). However, microbial features associated with sustained MRD negativity and diet which may impact microbiome and in turn MM outcomes have not been explored. Herein we evaluate the association of diet and microbiome with sustained MRD negativity in MM in the context of lenalidomide maintenance. MM patients eligible for lenalidomide maintenance during first-line therapy were enrolled (NCT02538198). MRD status was evaluated at baseline and annually with BM flow-cytometry (sensitivity >10-5). Sustained MRD negativity was defined as MRD negative for 1 year from enrollment. The relative abundance of butyrate producers and microbiome α-diversity were calculated from fecal 16S microbiome profiles at baseline, 3 months (m) and annually, along with quantification of fecal butyrate levels at 3m. Dietary patterns were collected using the Block Food Frequency Questionnaire (FFQ), for habitual diet and summarized via the Healthy Eating Index 2015 (HEI2015) scores in which higher scores imply healthier diets. The association between dietary and microbiome data were evaluated by Spearman’s rank correlation coefficient. Logistic regression was used to evaluate association between MRD status at 1 year (Y) and microbiome and dietary measurements. Statistical significance was defined using a two-sided significance level at 0.05. We had 49 patients with stool samples at 3m, 30 patients at Y1, 32 with stool butyrate levels at 3m, 59 with FFQ data, and the overlap between FFQ and stool samples was 34. The abundance of butyrate producers and/or butyrate levels were significantly associated with total HEI2015 and its components – total vegetables, dark green vegetables, legumes, whole grains, total protein, seafood and plant protein, fatty acids, refined grains, added sugars. Some aspects of healthier HEI score components (especially seafood and plant proteins) were associated with higher stool butyrate producers and butyrate levels at 3m, and sustained MRD negativity at Y1. Additionally, increased diversity, butyrate levels and butyrate producers at 3m was associated with increased MRD negativity at baseline and with sustained MRD negativity. In MM patients on lenalidomide maintenance, healthier diets (especially seafood and plant proteins) were associated with increased butyrate at 3m and in turn with higher MRD negativity. This is the first study to demonstrate an association between diet, microbiome, and sustained MRD negativity and provides a rationale for evaluating a targeted dietary intervention prospectively.