Abstract: Objective：To investigate the role of glycogen syntheses kinase 3β (GSK-3β) activation in response of colorectal cancer cells to epidermal growth factor receptor (EGFR) inhibitor gefitinib and insulin-like growth factor receptor-1 (IGFR-1) inhibitor AG1024. Methods：Western blot analysis was used to detect the expression levels of activated GSK-3β. Cell proliferation rate of colorectal cancer cells treated with GSK-3β inhibitor lithium chloride (LiCl) was determined by MTT assay. Laser-scaning microscopy based on immunofluorescence staining for activated GSK-3β performed to observe its cellular expression and intranuclear accumulation. Results：The expression level of activated GSK-3β significately increased in Lovo cells after treated with gefitinib, in contrast, no obvious changes in the other cell lines. HT29 cells showed a marked increase in GSK-3β activation after treated with the combination of gefitinib and AG1024, and so did HCT116 cells after the AG1024 treatment (P<0.05). Cell growth suppression of gefitinib-treated Lovo cells was rescued by the addition of LiCl, and the similar effect that resulted from LiCl action was also conducted with combination -treated HT29 cells, as well as AG1024-treated HCT116 cells (P<0.05). As confocal analysis shown, the significantly increased activated GSK-3β expression was found in the Lovo cells treated with the gefitinib, HT29 treated with the combination, and HCT116 treated with the AG1024, compared with their untreated-treated control groups respectively. More noticeable, subcellular localization of the activated GSK-3β displayed intranuclear accumulation when the cells were treated with the corresponding agents. Conclusion：Gefitinib and/or AG1024-induced cell growth suppression was mediated by GSK-3β activation, suggesting that the responses of colorectal cancer cells to EGFR/ IGFR-1β blockade could be predicted early in the course of treatment by measuring the activation of GSK-3β.