Although routine immunoprophylaxis has been known to reduce hepatitis B virus (HBV) transmission, immunoprophylaxis failure still occurs. The study aimed to investigate the protective efficacy of an improved immunoprophylaxis protocol to prevent mother-to-infant transmission of HBV and to explore the potential risk factors associated with immunoprophylaxis failure and low antibody response.A prospective observational cohort study was conducted from July 2012 to April 2015. A total of 863 HBsAg-positive mothers and their 871 infants (8 pairs of twins) were included in the study. Two different hepatitis B vaccine doses (20 or 10 μg) were administered to the infants based on the hepatitis B e-antigen (HBeAg) status of their mothers. Simultaneously, hepatitis B immunoglobulin (HBIG) was administered to the infants. Initial injections of HBIG and the hepatitis vaccine were given within 2 hours after birth. Rates of HBV infections among the infants were evaluated at 7 months of age. Factors associated with immunoprophylaxis failure and low responses to vaccination were analyzed by unconditional logistic regression..At 7 months of age, no immunoprophylaxis failure was observed in the 565 infants born to HBeAg-negative mothers. Among the 306 infants born to HBeAg-positive mothers, immunoprophylaxis failed in 16 infants (5.2%) of the infants and they were found to be HBsAg-positive. Further analysis showed that HBV DNA levels ≥10 IU/mL [odds ratio (OR) = 4.53, 95% confidence interval (95% CI): 1.19-17.34], delayed vaccination (OR = 4.14, 95% CI: 1.00-17.18), and inadequate initial injections (OR = 7.69, 95% CI: 1.71-34.59) were independently associated with immunoprophylaxis failure. Adequate titers of antibody to HBsAg (anti-HBs, ≥100 mIU/mL) were present in 96.5% of immunoprophylaxis-successful infants. For full-term infants, birth weights <3000 g were correlated with low immune responses to vaccination.This improved immunoprophylaxis protocol is effective in preventing perinatal transmission of HBV. Among infants with HBeAg-positive mothers, high HBV viral loads and inadequate and delayed initial injections were associated with immunoprophylaxis failure. The majority of the infants in our study produced adequate levels of protective anti-HBs titers after immunoprophylaxis. Additional efforts to further reduce perinatal transmission should be considered, especially for HBeAg-positive mothers.