This study aims to investigate the therapeutic effect of vitamin D (VD) combined with interferon (IFN) type I (IFN-alpha) on mice with hepatitis B and to explore the possible mechanism. The mice were divided into control group, model group, IFN-alpha group, and IFN-alpha(+) VD group. After 4 weeks, the mice of four groups were weighed, and the thymus and spleen indexes were calculated. The activity of alanine transaminase (ALT) and aspartate transaminase (AST) in the serum was detected while pathological changes of the liver were observed. The levels of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and interleukin-4 (IL-4) in serum were measured by enzyme-linked immunosorbent assay (ELISA). The proliferation of spleen lymphocytes was detected by MTT assay. Flow cytometry was used to detect the level of CD4(+), CD8(+), and CD4(+)/CD8(+) in peripheral blood. The levels of ALT and AST in serum were significantly lower in the IFN-alpha(+) VD group than those in the IFN-alpha group, but the thymus and spleen indexes were significantly higher. Although both IFN-alpha and IFN-alpha(+) VD can improve the damaged structure of live tissue, IFN-alpha(+) VD achieved higher efficacy than IFN-alpha alone. The serum IFN-gamma, TNF-alpha, and IL-2 levels were lower in the IFN-alpha(+) VD group compared with the IFN-alpha group, and no significant difference was found in IL-4 gamma Compared with the IFN-alpha group, the percentage of CD4(+) and the D4(+)/CD8(+) ratio were significantly increased, but the percentage of CD8(+) was reduced. The proliferation rate of splenic lymphocytes was higher in the IFN-alpha(+) VD group compared with the IFN-alpha group. IFN-alpha(+) VD was found to achieve higher efficacy than IFN-alpha alone for the treatment of hepatitis B in mice, possibly through increasing the immune level of mice.