Objective: To study the prevalence of rush poppers use among men who have sex with men (MSM) in Urumqi and to analyze the mediating effect of high-risk sexual behavior between the use of rush poppers and human papillomavirus (HPV) infection. Methods: From January to June 2018, 651 MSM were recruited through network and publicity. Data was collected by using online questionnaire and anal swab were collected from them for HPV genotyping. According to Baron and Kenny's criterion, multivariate logistic regression models was used to analyze the mediation effect of high-risks sexual behaviors (multiple sexual partners, anal intercourse condom use) between rush poppers use and HPV infection. Results: A total of 651 subjects were (32.0±8.0) years old, and 174 subjects (26.7%) had ever used rush poppers in the past 6 months, while 350 subjects (46.9%) had more than 2 sexual partners and 188 subjects (28.9%) did not use anal condom. After adjusting age and marital status, rush poppers use was associated with HPV infection (OR (95%CI) was 2.34 (1.63-3.36), P<0.05), and sexual partners in the past 6 months (OR (95%CI) was 2.72 (1.89-3.93), P<0.05).After adjustment for age, marital status, sexual partners in the past 6 months, and anal condom use, rush poppers use was still associated with HPV infection (OR (95%CI) was 2.21 (1.53-3.19), P<0.05).After adjustment and adjustment of age, marital status, rush poppers use and anal sex condom use, the number of sexual partners at nearly 6 months was still associated with HPV infection (OR (95%CI) was 1.46 (1.05-2.04), P<0.05). Conclusion Sexual partners in the past 6 months have a mediation effect between rush poppers use and HPV infection in Urumqi. For the prevention of sexually transmitted diseases, we should focus on rush poppers use.