Abstract Introduction The human microbiome has been implicated in a variety of clinical conditions. The semen harvests a compendium of microorganisms that potentially originate in different anatomic structures, such as the testis, prostate, and seminal vesicles. However, there is a paucity of data delineating the significance of the human microbiome in male reproductive health, specifically the testicular microbiome and its role over the seminal microbiota. Objective To analyze the changes in seminal microbiome after impeding the paracrine migration of testicular microbiome caused by undergoing a vasectomy. Methods Seminal samples were collected from 40 normozoospermic patients (normal concentration and motility according to the WHO parameters) and 36 post-vasectomy patients (samples were collected >3 months after vasectomy, the timeframe in which the microbiome is expected to have stabilized after the procedure). Sixteen patients gave a pre- and post-vasectomy semen sample. Richness (α-diversity) of the seminal microbiome, calculated by the number of operational taxonomic units, and clusterization (β-diversity), obtained by Bray Curtis distances, were compared between the normozoospermic and post-vasectomy SS, and then only among the paired SS. The relative abundance of species, genera, and phylum was compared among groups using analysis of compositions of microbiomes with bias correction (ANCOM-BC). Secondarily, the association between age, BMI, ethnicity, and recent antibiotic use (<3 months before obtaining SS) with seminal microbiome profiling was analyzed. Statistical significance was assessed at p<0.05. Results Operational taxonomic units (OTUs) were found to be higher in the normozoospermic group (median=35 OTUs) than in the post-vasectomy group (median=29 OTUs) (p=0.018). Similarly, in men who had paired semen samples, the pre-vasectomy samples (median=43 OTUs) had a higher α-diversity than the post-vasectomy samples (median=29 OUTs) (p<0.001), suggesting a higher community heterogenicity in the normozoospermic and pre-vasectomy groups. β-diversity was similar between normozoospermic and post-vasectomy groups (p=0.584), and also between paired semen samples (p=0.080) suggesting a similar community composition among groups. The relative abundance of species, genera, and phylum remained, in its majority, similar between groups. Nonetheless, when relative abundances with a prevalence of less than 10% were analyzed, we found that 12 species and 8 genera differed between normozoospermic and post-vasectomy samples. Age, BMI, ethnicity, and recent antibiotic use were not associated with seminal microbiome composition. Conclusions Our study highlights that the seminal microbiome without the paracrine contribution of testicular microbiome due to vasectomy, would exhibit fewer OTUs and a change in the relative abundance of species and genera. This causes a modification in α-diversity of the semen, implying that testicular microbiome contributes to the configuration and composition of the seminal microbiota. Further investigations are needed to determine the impact of these findings in male’s reproductive health. Disclosure Yes, this is sponsored by industry/sponsor: MicroGenDX Clarification Industry funding only - investigator initiated and executed study Any of the authors act as a consultant, employee or shareholder of an industry for: Acerus, Boston Scientific, Endo Pharmaceuticals and Coloplast.