Plasmid DNA vaccines have been widely explored for use in tuberculosis immunization but their immunogenicity needs improvement. In the present study, we incorporated the bovine herpesvirus 1 VP22 (BVP22)-encoding gene, which encodes a protein that demonstrates a capability for disseminating the expressed antigen to neighbouring cells, into a DNA vector in which it was fused to the Ag85B-encoding gene of Mycobacterium tuberculosis (Mtb), and investigated whether this linkage could enhance immune response and protective efficacy in C57BL/6 mice compared to plasmid DNA encoding Ag85B alone. After immunization in mice, Ag85B-specific ELISA antibodies and spleen lymphocyte proliferative responses induced by DNA co-expressing BVP22 and Ag85B were significantly higher than those obtained in mice immunized with Ag85B-encoding DNA alone, except for the number of gamma interferon secreting cells. In addition, based on histopathological examination and bacterial-load determination in lung and spleen, protection against intravenous Mtb H37Rv challenge evoked by the BVP22-Ag85B DNA immunization exceeded the response elicited by Ag85B DNA alone, which was not significantly different from that provided by Bacillus Calmette-Guérin (BCG). These results suggested that DNA vaccine consisting of BVP22 and Ag85B-encoding DNA enhanced immune response and protection against intravenous Mtb H37Rv challenge in mice, indicating that BVP22-encoding DNA might be a promising tool to enhance TB DNA vaccine efficacy.