Background <p>We aimed to elucidate the effects of rectal douching on gut microbial communities and their associated metabolites in men who have sex with men (MSM).</p> Methods <p>A community sample of HIV-uninfected MSM were recruited in Guangzhou, China. Participants were stratified into rectal douching and non-douching groups based on their rectal douching behavior within the past three months. Peripheral blood and fecal samples were collected. Serum markers of microbial translocation were quantified using enzyme-linked immunosorbent assay (ELISA). Gut microbiota composition was assessed via 16S rRNA gene sequencing of fecal samples, and microbial metabolites were profiled using a mass spectrometry-based platform.</p> Results <p>This study enrolls a total of 51 HIV-uninfected MSM (20 in the rectal douching group and 31 in the non-douching group). The two groups have comparable age distributions [median age: 25 years, interquartile range (IQR): 23-30 vs. 27 years, IQR: 24-31]. Significant structural shifts in microbial community composition are observed at both the phylum and genus levels in the rectal douching group relative to the non-douching group. The relative abundances of the genera <i>Clostridium</i>, <i>Lachnospira</i>, and <i>Turicibacter</i> are significantly lower in the rectal douching group compared to the non-douching group. Furthermore, rectal douching significantly alters a wide range of microbial metabolites. Notably, rectal douching appears to reduce gut barrier integrity, as evidenced by significantly elevated levels of the microbial translocation marker lipopolysaccharide-binding protein (LBP) in the rectal douching group.</p> Conclusions <p>Rectal douching among HIV-uninfected MSM is associated with gut microbiota dysbiosis, significant alterations in microbial metabolic profiles, and reduced gut barrier integrity. These findings underscore the need for increased awareness and health education within this population.</p>