To investigate the situation of recreational drug nitrous acid ester inhaler-rush poppers use in men who have sex with men (MSM). 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. 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 (2)= 14.879, =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 (=1.676, 95: 1.201-2.339, =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. Rush poppers use was related with HIV infection in MSM in Nanjing.