To evaluate the prevalence of human papillomavirus (HPV) sperm infection and its correlation with sperm parameters in patients who attended a fertility clinic. Cross-sectional clinical study. University-affiliated reproductive medicine clinic. A total of 308 male partners of couples undergoing in vitro fertilization techniques. Specimens of semen were collected from all patients. Sperm parameters were evaluated according to the World Health Organization manual. The presence of HPV DNA was researched by the combined use of two HPV assays and a highly sensitive nested polymerase chain reaction assay followed by HPV genotyping. To examine whether HPV was associated with the sperm, in situ hybridization (ISH) analysis was performed. Results of HPV investigation were compared with sperm parameters and ISH analysis. Twenty-four out of 308 semen samples (7.8%) were HPV DNA positive, but HPV infection did not seem to affect semen quality. Moreover, ISH revealed a clear HPV localization at the equatorial region of sperm head in infected samples. Oncogenic HPV genotypes were detected on spermatozoa from asymptomatic subjects, but a role of the infection in male infertility was not demonstrated.