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Background: Sexually transmitted infections (STIs) can be associated with infertility. Human papillomavirus (HPV) has been identified as a potential agent in male infertility. Also, anti-sperm antibodies (ASA) have been detected in men with infertility. The aim of this study was to investigate the prevalence and association of HPV and ASA in infected semen of infertile men., Methods: This cross-sectional study was performed on 96 infertile men referring to infertility treatment center of Kashan University of Medical Sciences during March 2017 till September 2017 in Iran. Semen analysis and diagnostic PCR test were performed for detection of HPV DNA. The semen parameters in HPV infected and ASA positive samples were compared with HPV non-infected and ASA negative samples. Chi square test was used to determine the correlation between variables and p<0.05 was considered statistically significant., Results: HPV DNA and ASA were detected in 17.4% and 15.2% of 96 semen samples, respectively. Semen volume, sperm count, sperm motility and the normal morphology rate were significantly decreased in HPV-positive subjects (p=0.004, p= 0.016, p<0.001, and p=0.017, respectively). Also, sperm motility was significantly decreased in ASA-positive subjects (p=0.002), also patients with HPV infection had a higher rate of ASA than the non-HPV group. In contrast to ASA, HPV infection had a significant correlation with education level (p=0.039)., Conclusion: The findings suggest that asymptomatic seminal infection of HPV and ASA by adversely affecting sperm quality, in particular sperm motility and count, may play an important role in male infertility., Competing Interests: Conflict of Interest The authors declare that there is no conflict of interest., (Copyright© 2020, Avicenna Research Institute.)