UNLABELLED: Gut microbiome of men who have sex with men (MSM) exhibits distinctive characteristics compared with general populations. The dysbiosis of the gut microbiome in MSM is also associated with the onset and evolution of HIV infection. Enterotype is an important feature of the gut microbiome and remains unaffected by demographic factors. However, the enterotypes of gut microbiome in MSM are unclear. The associations between enterotypes and high-risk sexual behaviors in this population also remain to be elucidated. HIV-negative MSM were recruited in this study. Fecal samples of the participants were collected and subjected to 16S rRNA gene sequencing. Enterotype clusters were determined by Jensen-Shannon divergence based on genus-level relative abundance. Microbial function predictions were conducted by PICRUSt2 software. Univariate and multivariate logistic regression approaches were utilized to analyze the associations of enterotypes with sexual behaviors. A three-category random forest machine learning model was performed to further examine the correlation between abundant microbiome in each enterotype cluster and anal sex roles. Two enterotype clusters were identified in our data sets, primarily driven by genera and . The alpha diversity was comparable between the two enterotype clusters. Microbial metabolic functions significantly differed, and multivariate logistic regression indicated a significant association between anal sex role and enterotype. The results of the three-category random forest model indicate that the dominant bacterial communities in gut enterotypes can effectively differentiate MSM who engage exclusively in receptive anal intercourse from those who engage in insertive or versatile anal intercourse (AUC: 0.6400, 0.6929, respectively). We identified two enterotype clusters of gut microbiome in HIV-negative MSM. Enterotypes of MSM were significantly associated with anal sex roles. These findings further highlight the close correlation between the gut microbiome and anal intercourse roles. IMPORTANCE: Our study's discovery that gut microbiome enterotypes are significantly associated with anal sex roles in HIV-negative MSM opens a new frontier in understanding the complex interplay between microbiology and sexual health. This finding underscores the urgency of delving into the mechanistic connections between the gut microbiome, sexual behaviors, and HIV infection. By identifying modifiable factors influencing gut microbiome composition, we have paved the way for developing personalized preventive strategies that could disrupt the transmission dynamics of HIV within this high-risk population. This research contributes to the fundamental understanding of the gut microbiome's role in the sexual health of MSM, making it a pivotal advancement in the fields of gut microbiome research and sexual health.