Abstract Dysbiosis of the microbiome is an important factor in susceptibility to diseases such as inflammatory bowel disease (IBD). Vitamin D has known immunoregulatory roles and decreased levels of vitamin D are linked to IBD. Mice lacking the enzyme 1-α-hydroxylase (Cyp27B1 KO) cannot metabolize 25(OH)D3 into bio-active 1,25(OH)2D3. These mice are more susceptible to experimental colitis compared to WT controls. Here we wanted to determine the effect of vitamin D3 on the urinary metabolome, since microbial metabolites can be detected in urine. Urine was collected from WT, WT+20,000IU vitamin D3, and WT and Cyp27B1 KO littermates. Urine samples were profiled by ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) and multivariate data analysis tools. WT mice supplemented with vitamin D and WT and Cyp27B1 KO mice displayed changes in microbial metabolites compared to unsupplemented mice. Metabolites associated with the gut microbiota: p-cresol sulfate, indoxyl sulfate, xanthurenic acid, and betaine were different as a function of vitamin D3 supplementation or Cyp27B1 expression. Alterations in the urinary metabolome suggest a role for vitamin D3 in regulating the microbiota. Metabolite analysis will aid our understanding of the mechanisms by which vitamin D3 affects host health and the microbiota, and future work will address how changes in the microbiota affect susceptibility to experimental IBD.