Background: Approximately half a million vasectomies are performed every year in the USA. There is a paucity of literature on the impact of male sterilization on the semen microbiome and whether it prompts microbiota dysbiosis., Objective: To investigate if vasectomy induces changes in the seminal microbiome via comparison of semen samples from men before and after vasectomy, and if the seminal microbiome profiles for vasectomized men follow a particular pattern with respect to diversity and abundance., Design, Setting, and Participants: From July 2021 to February 2022, we prospectively collected and analyzed semen samples from 58 men at one outpatient clinic. Eighteen men provided a semen sample before and 3 mo after vasectomy. We also collected semen samples from 22 fertile nonvasectomized men and from a further 18 vasectomized men at 3 mo after vasectomy., Outcome Measurements and Statistical Analysis: Semen microbiome α-diversity, beta-diversity, and relative abundance were compared initially between paired and then between unpaired vasectomized and nonvasectomized samples. Analysis of variance (ANOVA), permutational multivariate ANOVA, and analysis of the composition of microbiomes with bias correction were used to assess differences., Results and Limitations: In both paired and unpaired sets of samples, a decreasing trend for α-diversity in semen after vasectomy was observed. Shannon diversity, the relative abundance of species with an abundance >2%, and composition were not significantly changed. Sphingomonas, Brevundimonas, and Paracoccus abundance decreased after vasectomy, while Corynebacterium abundance increased. The results may be limited by the sample size and lack of demographic heterogeneity., Conclusions: Vasectomy is followed by a decrease in α-diversity and changes in the relative abundance of bacterial species in the semen microbiome. Further investigation is necessary to understand the clinical significance of these changes after vasectomy., Patient Summary: We evaluated changes in the bacteria species in semen after vasectomy. We found that vasectomy decreased the richness and evenness of bacteria species in semen, but the overall bacterial community remained similar. Further studies are needed to assess the implications of changes in semen bacteria after vasectomy., (Copyright © 2022 European Association of Urology. Published by Elsevier B.V. All rights reserved.)