Rituximab efficacy in cancer therapy depends in part on induction of complement-dependent cytotoxicity (CDC). Human CD59 (hCD59) is a key complement regulatory protein that restricts the formation of the membrane attack complex, thereby inhibiting induction of CDC. hCD59 is highly expressed in B-cell non–Hodgkin's lymphoma (NHL), and upregulation of hCD59 is an important determinant of the sensitivity of NHL cells to rituximab treatment. Here, we report that the potent hCD59 inhibitor rILYd4 enhances CDC in vitro and in vivo, thereby sensitizing rituximab-resistant lymphoma cells and primary chronic lymphocytic leukemia cells (CLL) to rituximab treatment. By defining pharmcokinetic/pharmacodynamic profiles of rILYd4 in mice, we showed that by itself rILYd4 does not adversely mediate in vivo hemolysis of hCD59-expressing erythrocytes. Increasing expression levels of the complement regulators CD59 and CD55 in rituximab-resistant cells occur due to selection of preexisting clones rather than de novo induction of these proteins. Moreover, lymphoma cells overexpressing CD59 were directly responsible for the resistance to rituximab-mediated CDC therapy. Our results rationalize the use of rILYd4 as a therapeutic adjuvant for rituximab treatment of rituximab-resistant lymphoma and CLL. Furthermore, they suggest that preemptive elimination of CD59-overexpressing subpopulations along with rituximab treatment may be a useful approach to ablate or conquer rituximab resistance. Cancer Res; 71(6); 2298–307. ©2011 AACR.