Abstract Purpose: Evasion from chemotherapy-induced apoptosis due to p53 loss strongly contributes to drug resistance. Identification of specific targets for the treatment of drug-resistant p53-null tumors would therefore increase the effectiveness of cancer therapy. Experimental Design: By using a kinase-directed short hairpin RNA library and HCT116p53KO drug-resistant colon carcinoma cells, glycogen synthase kinase 3 beta (GSK3B) was identified as a target whose silencing bypasses drug resistance due to loss of p53. p53-null colon cancer cell lines with different sets of mutations were used to validate the role of GSK3B in sustaining resistance and to characterize cell death mechanisms triggered by chemotherapy when GSK3B is silenced. In vivo xenograft studies were conducted to confirm resensitization of drug-resistant cells to chemotherapy upon GSK3 inhibition. Colon cancer samples from a cohort of 50 chemotherapy-treated stage II patients were analyzed for active GSK3B expression. Results: Downregulation of GSK3B in various drug-resistant p53-null colon cancer cell lines abolished cell viability and colony growth after drug addition without affecting cell proliferation or cell cycle in untreated cells. Cell death of 5-fluorouracil (5FU)–treated p53-null GSK3B-silenced colon carcinoma cells occurred via PARP1-dependent and AIF-mediated but RIP1-independent necroptosis. In vivo studies showed that drug-resistant xenograft tumor mass was significantly reduced only when 5FU was given after GSK3B inhibition. Tissue microarray analysis of colon carcinoma samples from 5FU-treated patients revealed that GSK3B is significantly more activated in drug-resistant versus responsive patients. Conclusions: Targeting GSK3B, in combination with chemotherapy, may represent a novel strategy for the treatment of chemotherapy-resistant tumors. Clin Cancer Res; 19(14); 3820–31. ©2013 AACR.