Simple SummaryThis study focused on the relationship between microRNA-23a/b-3p and genes involved in human spermatogenesis, with the aim of understanding how this microRNA targets these genes and how it affects male fertility. Through computational prediction and experimental assays, we identified specific genes that are targeted by microRNA-23a/b-3p. By intentionally modifying the binding sites of microRNA-23a/b-3p in these genes, we confirmed the direct targeting. To validate their findings, we analyzed sperm samples from men with low sperm count and motility compared to a control group. The results revealed that the men with fertility issues had lower expression levels of the target genes. Furthermore, we found a positive correlation between basic semen parameters and the reduced expression levels of the target genes. This indicates that microRNA-23a/b-3p plays a significant role in spermatogenesis by regulating the expression of target genes associated with male fertility problems, and it also has an impact on basic semen parameters. Overall, this study provides important insights into the involvement of microRNA-23a/b-3p in spermatogenesis and its potential influence on male fertility.AbstractThe expression levels of various genes involved in human spermatogenesis are influenced by microRNAs (miRNAs), specifically microRNA-23a/b-3p. While certain genes are essential for spermatogenesis and male germ cell function, the regulation of their expression remains unclear. This study aimed to investigate whether microRNA-23a/b-3p targets genes involved in spermatogenesis and the impact of this targeting on the expression levels of these genes in males with impaired fertility. In-silico prediction and dual-luciferase assays were used to determine the potential connections between microRNA-23a/b-3p overexpression and reduced expression levels of 16 target genes. Reverse transcription-quantitative PCR (RT-qPCR) was conducted on 41 oligoasthenozoospermic men receiving infertility treatment and 41 age-matched normozoospermic individuals to verify the lower expression level of target genes. By employing dual-luciferase assays, microRNA-23a-3p was found to directly target eight genes, namelyNOL4, SOX6, GOLGA6C, PCDHA9, G2E3, ZNF695, CEP41, andRGPD1, while microRNA-23b-3p directly targeted three genes, namely SOX6,GOLGA6C, andZNF695. The intentional alteration of the microRNA-23a/b binding site within the 3′ untranslated regions (3′UTRs) of the eight genes resulted in the loss of responsiveness to microRNA-23a/b-3p. This confirmed thatNOL4, SOX6, GOLGA6C, PCDHA9, andCEP41are direct targets for microRNA-23a-3p, whileNOL4, SOX6, andPCDHA9are direct targets for microRNA-23b-3p. The sperm samples of oligoasthenozoospermic men had lower expression levels of target genes than age-matched normozoospermic men. Correlation analysis indicated a positive correlation between basic semen parameters and lower expression levels of target genes. The study suggests that microRNA-23a/b-3p plays a significant role in spermatogenesis by controlling the expression of target genes linked to males with impaired fertility and has an impact on basic semen parameters.