The implications of vitamin D deficiency on the immune system have become clearer in recent years, being associated with less immune response following HBV vaccine. We aimed to elucidate the effect of vitamin D supplementation and UVB exposure on short- and long-term performance of hepatitis B vaccine. Forty-five male rabbits were randomly divided into 3 groups that were immunized with recombinant HBsAg. The first group (group I) represented a negative control group, whereas group III rabbits were administered with commercially available 1,25 (OH)2 vitamin D as an alternative for UVB exposure in group II. Results showed that vitamin D concentrations were significantly higher in UVB exposed group compared to both negative control and vitamin D-supplemented groups during short- and long-time intervals. In addition, means of anti-HBsAg isotypes’ levels and anti-HBsAg IgG avidity% were significantly higher in negative control group compared to other groups during short- and long-time intervals. Moreover, vitamin D serum concentration was positively correlated with anti-HBsAg IgG level and avidity % in both negative control and vitamin D-supplemented groups, while it was negatively correlated with anti-HBsAg IgM level in negative control group. It can be concluded from the above results that UVB radiation may have both augmenting and suppressive effects and that circulating serum vitamin D concentration may have a positive association with premium immune modulation following HBV vaccination.