To evaluate the impact of the universal infant HepB vaccination program on hepatitis B virus infection in Hangzhou, China. Hepatitis B incidences and HepB vaccination rates of Hangzhou from 1990 to 2003 were acquired from the historical paper-documents, while which were derived from China Information System for Diseases Control and Prevention and Zhejiang Information System for Immunization Program respectively from 2004 to 2013. A serology survey among person aged 0-59 y was conducted in Hangzhou in 2006. Participants were selected by stratified, multi-stage random sampling. Serum specimens were tested for HBsAg, anti-HBs , anti-HBc , HBeAg and anti-HBe by ELISA. For the past 24years, hepatitis B incidence and mortality of Hangzhou declined dramatically ((2) = 3.2 x 10(4); (2) = 172.443; both P for trend < 0.001). Both urban and rural incidence descended ((2)(urban) = 1.904 x 10(4); (2)(rural) = 1.633 x 10(4) ; both P for trend < 0.001).Hepatitis B patients mainly concentrated in 20-40 y old; workers and farmers were the main infection occupations, which was varies in different years ((2) = 1.619 x 10(3), P < 0.001). Significant association was found between incidence of hepatitis B and HepB vaccination rate (r = 0 .946, (2) = 11.813, P-for trend = 0.001). A total of 5605 participants aged 0-59 y included in this serological survey. The prevalence of HBsAg, anti-HBs, anti-HBc, HBeAg and anti-HBe were 6.19%, 45.83%, 57.25%, 0.62%, and 4.37%, respectively. Hangzhou has successfully integrated the HepB into routine immunization programs and this has had a significant impact on decreasing the incidence of hepatitis B infection.