The chimeric antiganglioside GM2 monoclonal antibody (MAb) KM966, which showed high effector functions such as complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), potently suppressed growth and metastases of GM2-positive human cancer cells inoculated into mice. To further improve the therapeutic efficacy of the anti-GM2 MAb in humans, we constructed a humanized anti-GM2 MAb, KM8969. The humanized KM8969 was more efficient in supporting ADCC against GM2-positive human cancer cell lines than the chimeric KM966, whereas complement-dependent cytotoxicity was slightly reduced in the humanized KM8969. In addition, the humanized KM8969 was shown to exert potent ADCC mediated by both lymphocytes and monocytes. To investigate the effect of the humanized KM8969 on the biological function of GM2 in the condition physiologically mimicking formation and growth of cancer masses, the heterospheroids composed of normal human dermal fibroblasts and GM2-positive human lung cancer cells were developed. Interestingly, the humanized KM8969 gave rise to growth inhibition of heterospheroids without dependence of the effector functions. Morphological and immunocytochemical analysis suggested that the inhibitory effect was due to the apoptosis of GM2-positive cancer cells in the heterospheroids. The result indicates that GM2 captured by the antibody on the cell surface loses its physiological function that plays a critical role in maintaining the three-dimensional growth of cancer cells in contact with its own cells or other type of cells in a microenvironment. The humanized KM8969, which can destroy the cancer cells via blocking functional GM2 on the cell surface as well as the effector functions, would have extraordinary potential in human cancer therapy.