OBJECTIVE Is the presence of human papillomavirus (HPV) in semen associated with impairment of semen quality? CONCLUSIONS In a large cohort of males seeking fertility evaluation, no associations were observed between seminal HPV presence and semen parameters. BACKGROUND HPV is commonly detected in semen samples. Whether the presence of HPV is related to impairment of semen quality, remains unclear. METHODS This cross-sectional study included a cohort of 430 males. METHODS Male partners in couples seeking fertility evaluation provided one semen sample per person. Semen samples were tested for HPV-DNA using GP5+/6+-PCR. Sperm concentration was counted and motility was assessed in a Makler counting chamber at a magnification of ×200. The presence of antisperm antibodies was assessed by a mixed agglutination reaction (MAR)-test. RESULTS Overall HPV was detected in 14.9% (64/430) of semen samples, including 2.1% (9/430) that contained both high-risk (hr) HPV and low-risk (lr) HPV types, 8.8% (38/430) with exclusively hrHPV types and 4.0% (17/430) with exclusively lrHPV types. The presence of HPV in semen was not associated with the age of the participants, seminal pH, semen volume, total sperm count, sperm concentration, progressive motility or the presence of antisperm antibodies. CONCLUSIONS This study did not observe an association between HPV presence in semen and impairment of semen quality. However, we cannot exclude an effect of seminal HPV on early embryo development and clinical reproductive outcomes. CONCLUSIONS As HPV is frequently present in semen, screening of donor semen for HPV should be considered to prevent iatrogenic cervical HPV infections in the recipient. However our findings do not support standardized HPV testing of semen in the diagnostic work-up of subfertile couples. BACKGROUND This study was sponsored by an unrestricted grant of Stichting Researchfonds Pathology Amsterdam, the Netherlands. P.J.F.S. has been on the speakers bureau of Roche, Gen-Probe, Abbott, Qiagen and Seegene and has been a consultant for Crucell B.V. J.B. has been on the speakers bureau of Qiagen and has been a consultant for Roche, DDL Diagnostic Laboratory, GlaxoSmithKline and Merck. D.A.M.H. has been member of the scientific advisory boards of Amgen and Pfizer, and has been on the speakers bureau of Hologic/Gen-Probe. C.J.L.M.M. has been on the speakers bureau of GlaxoSmithKline, Qiagen, Merck, Roche, Menarini and Seegene, has served occasionally on the scientific advisory board of GlaxoSmithKline, Qiagen, Merck, Roche and Genticel, and has occasionally been a consultant for Qiagen. Formerly, C.J.L.M.M. was a minority shareholder of Delphi Biosciences, which bankrupted in 2014. C.J.L.M.M. is a minority shareholder of Diassay B.V. P.J.F.S., D.A.M.H. and C.J.L.M.M. have minority stake in Self-Screen B.V., a spin-off company of VU University Medical Center. R.L., M.G.D., P.G.A.H., D.T.M.P., and I.H. do not have any conflicts of interest to disclose. BACKGROUND Not applicable.