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Introduction: In China, the prevalence and incidence of HIV among men who have sex with men (MSM) in large‐sized cities have drawn much attention. In contrast, there has been a paucity of research focussing on the sexual health of MSM of medium‐sized cities. This study fills this important gap in the knowledge by investigating the sexual health of MSM in a medium‐sized city (Yangzhou) and a large‐sized city (Guangzhou). Methods: A baseline survey and a prospective cohort study were conducted among MSM in Yangzhou and Guangzhou from July 2009 to September 2010. A total of 622 MSM (317 from Yangzhou and 305 from Guangzhou) were screened for eligibility. Prevalence and incidence of HIV infection, as well as its risk factors, were investigated. Results: Baseline HIV prevalence was 14.5%, and overall HIV incidence density was 6.78 per 100 person‐years (PY) among Yangzhou MSM. Risk factors for HIV prevalence that were significant in multivariate models were older age, married status, unprotected sex with female partners, sexually transmitted disease (STD)‐associated symptoms and syphilis positivity. Risk factors for HIV incidence that were significant in multivariate models were STD‐associated symptom and syphilis positivity. Compared to Yangzhou MSM, Guangzhou MSM had a lower HIV prevalence (6.2%; p Conclusions: This study showed a high prevalence and incidence of HIV among Yangzhou MSM, which suggest a more serious HIV epidemic than that in large‐sized cities. Further investigation targeting MSM in medium‐sized cites is urgently needed to prevent the spread of the HIV epidemic in China., Introduction In China, an estimated 780,000 people were living with HIV at the end of 2011, according to the latest estimation conducted jointly by the Ministry of Health of China, [...]