Background: Accidental exposure to sharp instruments is an important problem for health care students. Thus, the aim of this study was to determine the rate of immunity in health care students 2 decades after national neonatal hepatitis B (HB) vaccination. Methods: All junior students attending medicine, nursing and midwifery schools were screened for anti-HBs. One dose of hepatitis B vaccine was offered to all participants who did not have antibodies to HB surface antigen (anti-HBs) of > 10 IU/L; then, they were tested for anti-HBs after a month. The participants were classified into 3 groups: postboosting nonimmune, postboosting immune, and preboosting immune. Chi square test and ANOVA were used for data analysis. Results: In the first step, 65.20% of participants did not show immunity, but after receiving a booster dose, only 6.0% remained nonimmune. The mean age of nonimmune students was significantly higher than that of students who had postboosting immune and preboosting immune status (p=0.001 and 0.002, respectively). Also, the mean injection time from last shot was higher in postboosting immune group compared to preboosting immune group (p<0.001). Also, prebooster anti-HBs level was significantly different among participants with suboptimal response and those who developed anamnestic response, indicating preserved immune memory (p=0.001). Conclusion: High anamnestic response to HBV booster dose indicates sufficient immunity to HBV in the majority of health care students. However, identifying students who cannot respond to a booster dose of vaccine seems to be necessary at the beginning of health care courses.