Background: This was a cross-sectional study in China, we analyzed the levels of macrophages(Mφ) in semen. The study evaluated the influence of the levels of Mφ in semen on sperm quality.Methods: The subjects were 78 males between 25 and 35 years old. The samples were divided into a low group(Mφ<6×105/ml) and a high group (Mφ>6×105/ml). Evaluation included consideration of the influencing factors of male semen quality, macrophage concentration, sperm motility, morphology, membrane integrity DNA fragmentation index (DFI), anti-sperm antibodies (AsAb), IL-10, and IL-12 in semen.Results: There was no difference in the physical or chemical indices of the semen, sperm concentration, AsAb, IL-10, or IL-12 between the two groups (P>0.05). The percentage of sperm forward motility (PR%), the rate of normal sperm shape, and the integrity of cell membranes in the low group were higher than those in the high group (P<0.05), while the percentage of sperm inactivity (IM%), the rate of sperm head deformity, the rate of deformity in the neck and middle segment, the sperm malformation index (SDI), the abnormal sperm index (TZI), and the sperm DFI in the low group were lower than those in the high group (P<0.05). The concentration of Mφ in the semen was linearly correlated with sperm concentration, sperm PR%, IM%, sperm normal shape rate, head deformity rate, neck and middle deformity rate, SDI, TZI, sperm DFI, and sperm cell membrane integrity (P<0.05), but there was no linear correlation with IL-10 or IL-12 (P>0.05).Conclusions: The concentration of Mφ in semen had no significant correlation with semen volume or sperm concentration, but it did have a significant negative correlation with sperm motility, sperm morphology, cell membrane integrity, and DNA breakage rate. There was no significant correlation with the concentration of IL-10 or IL-12.