<b><i>Background:</i></b> Sexually transmitted infections are a major cause of infertility. Human papillomavirus (HPV) infection is one of the most common viral infections of the female genital tract. Only a limited number of studies have investigated the influence of HPV on fertility and its impact remains controversial. <b><i>Objective:</i></b> We investigated the relationship between cervical HPV infection and pregnancy outcome after intrauterine insemination (IUI). Since other sexually transmitted infections could also influence outcome, we also analyzed the influence of <i>Trichomonas vaginalis</i> (TV) and <i>Chlamydia trachomatis</i> (CT) on pregnancy outcome. <b><i>Methods:</i></b> We performed a retrospective analysis of 590 women who underwent 1,529 IUI cycles at AML between 2010 and 2014. Positivity of 18 different HPV types (6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68) and TV was assessed by PCR in cervical cytology specimens. CT status was ascertained by detection of IgA/IgG antibodies on serum samples or by PCR on cervical swabs. <b><i>Results:</i></b> The HPV prevalence per IUI cycle was 11.0 and 6.9% for CT; none of the women tested positive for TV. HPV-positive women were six times less likely to become pregnant after IUI (1.87 vs. 11.36%; p = 0.0041). There was no significant difference in pregnancy rates between women with or without a history of CT (8.51 vs. 11.10%; p > 0.05). <b><i>Conclusion:</i></b> Detection of HPV is associated with a negative IUI outcome.