Objective: To investigate the situation of recreational drug nitrous acid ester inhaler-rush poppers use in men who have sex with men (MSM). Methods: From April to June in 2014, October to December in 2014, April to June in 2015, three round cross-sectional surveys were conducted in MSM recruited in Nanjing by means of serum test and questionnaire survey. Results: Of the 1 721 MSM surveyed in three round, 19.3% (332/1 721) had ever used rush poppers, the proportions of MSM who used rush poppers in three round surveys showed an increasing trend (liner by liner χ(2)= 14.879, P=0.000), which was 12.6% (86/681), 27.6% (121/439) and 20.8% (125/601) respectively. Rush poppers use was associated with HIV infection significantly (OR=1.676, 95%CI: 1.201-2.339, P=0.002). Compared with MSM without rush poppers use, the MSM with rush poppers use were mainly aged <30 years, unmarried, received college and higher education, had lived in Nanjing for less than 2 years, seek sex partners through internet and identified themselves as homosexual orientation. Multivariate logistic analysis showed that less than 30 years old, living in Nanjing for less than 2 years, with college and higher education level, more than 2 sex partners in the past 6 months, being diagnosed with STDs in the previous one year and using traditional drugs were the risk factors associated with rush poppers use. Conclusion: Rush poppers use was related with HIV infection in MSM in Nanjing.