Mucin 1 (MUC1) is a tumor antigen, and the most important epitopes that can induce cytotoxic T lymphocytes (CTL) reside in the variable-number tandem repeats (VNTR). Heat shock protein (HSP) complexes isolated from tumors have been shown to induce specific anti-tumor immunity. HSP alone can also induce nonspecific immunity. To explore the possibility to utilize the specific anti-tumor immunity induced by MUC1 VNTR and the nonspecific immunity induced by HSP, we constructed a recombinant protein (HSP65-MUC1) by fusing Bacillus Calmette-Guérin-derived HSP65 with the MUC1 VNTR peptide and tested its ability to induce anti-tumor activities in a tumor challenge model. The growth of MUC1-expressing tumors was significantly inhibited in mice immunized with HSP65-MUC1, both before and after tumor challenge. A much larger percentage of immunized mice survived the tumor challenge than non-immunized mice. Correlating with the anti-tumor activity, HSP65-MUC1 was shown to induce MUC1-specific CTL as well as nonspecific anti-tumor immunity. In the human system, HSP65-MUC1-loaded human DC induced the generation of autologous MUC1-specific CTL in vitro. These results suggest that exogenously applied HSP65-MUC1 may be used to treat MUC1 tumors by inducing the epitope-specific CTL as well as nonspecific anti-tumor responses mediated by the HSP part of the fusion protein.