Attenuated strains of bacteria have been developed as potential live vectors to express homologous or heterologous antigens of many pathogens for inducing protective immune responses. The non-pathogenic and rapidly growing Mycobacterium smegmatis can be transformed effectively by genes for pathogenic antigens, and has been used as a valuable vector for the development of live vaccines. However, little is known on whether M. smegmatis could be transformed with the genes for HBV antigens and could express those genes, and whether vaccination with recombinant M. smegmatis could induce humoral and cellular immune responses in vivo. Both the core protein and preS1 peptide of the hepatitis B virus (HBV) are immunogenic and can induce cellular and humoral immune responses. This made them ideal platform for the development of new vaccines. In the present study, both recombinant M. smegmatis and DNA vaccines were generated to express the CS1 antigen, a fusion protein that comprises truncated core protein (amino acids 1-155) and preS1 peptide (amino acids 1-55) of HBV. Following vaccination of BALB/c mice with the live recombinant M. smegmatis, the CS1-based DNA vaccine, or controls, antigen-specific humoral and cellular immune responses were characterized. Vaccination with live recombinant M. smegmatis induced a stronger cellular immune response and a longer period of humoral immune response than with the DNA vaccination. These results indicate that the recombinant M. smegmatis can express efficiently immunogenic CS1 antigen of HBV in vivo, and may be used for the prevention of HBV infection.