Trastuzumab, an anti-HER2 monoclonal antibody, is the mainstay treatment for of HER2-positive breast cancer. However, trastuzumab resistance is often observed during treatment. Therefore, new therapeutic strategies are needed to enhance the clinical benefits of trastuzumab. Maitake β-glucan MD-Fraction, isolated from Grifola frondosa, inhibits tumor growth by enhancing immune responses. In this study, we examined the effect of MD-Fraction on trastuzumab treatment of HER2-positive breast cancer. MD-Fraction did not directly inhibit the survival of HER2-positive breast cancer cells, alone or in the presence of trastuzumab in vitro. In HER2-positive xenograft models, the combination of MD-Fraction and trastuzumab was more effective than trastuzumab alone. Peripheral blood lymphocytes and splenic natural killer cells isolated from BALB/c nu/nu mice treated with MD-Fraction showed enhanced trastuzumab-induced antibody-dependent cellular cytotoxicity (ADCC) ex vivo. MD-Fraction-treated macrophages and neutrophils did not show enhanced trastuzumab cytotoxicity in the presence of heat-inactivated serum, but they showed enhanced cytotoxicity in the presence of native serum. These results suggest that MD-Fraction-treated macrophages and neutrophils enhance trastuzumab-induced complement-dependent cellular cytotoxicity (CDCC). Treatment of HER2-positive breast cancer cells with MD-Fraction in the presence of trastuzumab and native serum increased C3a release and tumor cell lysis in a dose-dependent manner, indicating that MD-Fraction enhanced trastuzumab-induced complement-dependent cytotoxicity (CDC) by activating the complement system. This study demonstrates that the combination of trastuzumab and MD-Fraction exerts a greater antitumor effect than trastuzumab alone by enhancing ADCC, CDCC, and CDC in HER2-positive breast cancer.