One of the major issues in current antibody therapy is insufficient efficacy. Various biological factors relating to the host's immune system or tumor cells have been suggested to reduce the efficacy of anti-CD20 therapy in B-cell malignancies. In this study, we characterized the in vitro anti-lymphoma activity of anti-CD20 antibodies having a novel engineered heavy chain with enhanced complement-dependent cytotoxicity (CDC). Anti-CD20 antibodies having a variant heavy constant region of mixed IgG1/IgG3 isotype, which have previously been found to enhance CDC, were investigated for their in vitro CDC against lymphoma cells and whole blood B-cell depletion activity. Use of the variant constant region greatly increased the CDC of an anti-CD20 antibody having variable regions identical to those of rituximab to the level shown by an IgG1 antibody of ofatumumab. Although the whole blood assay showed different cytotoxicity patterns among individual blood donors, the CDC-enhancing variant of rituximab showed higher activity than the parent IgG1 and consistently showed maximized activity when further combined with antibody-dependent cellular cytotoxicity (ADCC)-enhancing modification by fucose removal from Fc-linked oligosaccharides. In addition, the rituximab variant showed potent CDC against transfectant cells with lower CD20 expression and chronic lymphocytic leukemia-derived cell lines with higher complement regulatory proteins. These findings suggest that CDC enhancement, both alone and in combination with ADCC enhancement, increases the anti-lymphoma activity of anti-CD20 antibodies irrespective of individual differences in effector functions, and renders current anti-CD20 therapy capable of overcoming the potential resistance mechanisms.