Abstract Background and Aims Kidney graft rejection still represents the major cause of graft loss in kidney transplant recipients. Of growing interest is the bidirectional relationship between gut microbiome and immune system suggesting that gut microbiota can affect allograft outcome. Method In this cross-sectional case control study, we characterized the gut microbial profile of adult renal transplant recipients with and without graft rejection in order to define a cohort-specific microbial fingerprint through 16S rRNA gene sequencing. We used very strict inclusion and exclusion criteria to address confounder of microbiota composition. Results Different relative abundances in several gut microbial taxa were detectable in control patients compared to patients with kidney allograft rejection. Alpha diversity was lower in the rejection group and beta diversity revealed dissimilarity between patients with and without kidney graft rejection (P < .01). When the rejection group was stratified according to different types of allograft rejection, major changes were identified between patients with chronic T cell mediated rejection (cTCMR) and controls. Changes in alpha diversity within the gut microbiome were related to the probability of cTCMR (P < .05). Kidney transplant patients without rejection showed significant enrichment of rather anti-inflammatory taxa whereas in the rejection group bacteria well known for their role in chronic inflammation were increased. For example, ASV 362 belonging to the genus Bacteroides and ASV 312 belonging to Tanerrelaceae were enriched in no rejection (P < .001 and P < .01) whereas ASV 365 was enriched in patients with allograft rejection (P = .04). Looking at metagenomic functions, a higher abundance of genes coding for enzymes involved in bacterial multidrug resistance and processing of short chain fatty acids was found in patients without rejection but an increase in enzymes involved in NADPH production was seen in patients with allograft rejection. Conclusion A distinct microbial fingerprint of patients with allograft rejection might serve as non-invasive biomarker in the future.