Abstract Maltose binding protein (MBP) is a component of the maltose transport system in the periplasm of Escherichia coli. It is commonly believed that MBP has minimal effects on the bioactivity, thus, it is widely used in the purification of recombinant proteins. Here, we found that the combined immunization with MBP and Bacillus Calmette–Guerin (BCG) significantly inhibited tumor growth compared with MBP or BCG immunization alone in a mouse lung carcinoma model. Further studies showed that MBP nonspecifically activated T helper 1 (Th1) cells and enhanced the BCG-induced Th1 cell activation. Moreover, MBP or BCG immunization alone increased the activities of natural killer (NK) cells and macrophages, and the combined immunization with MBP and BCG induced a synergistic effect on the activities of NK cells and macrophages. These results suggest that MBP possesses potent immune enhancement activities, and that the combination of MBP and BCG-induced synergistic antitumor effects might be mediated mainly through the activation of Th1 cells, NK cells and macrophages.