The work is to determine the effects of γ-irradiation on GSK3 β coupled signaling transduction pathway in IEC-6 cells, and protect effects of regulation of GSK3 β and caspase-3 activation onγirradiation induced IEC-6 cells injuries. The changes of GSK-3 β and caspase-3 mRNA expression were determined by RT-PCR. The change of GSK-3 β phosphorylation was determined by western blotting. The changes of GSK-3 β enzymes activity were tested by radiation activity measurement. The changes of caspase-3 enzymes activity were assessed via a colorimetric assay with specific substrates. The IEC-6 cells apoptosis were determined by Hoechst 33342 staining and DNA ladder. The results show that 6Gy γ-irradiation increased apoptotic cell percentage in IEC-6 cells. Significant changes of GSK3 β and downstream signal transduction molecular caspase-3 in mRNA expression, GSK3 β protein dephosphorylation and protein enzymatic activation were observed after the irradiation. GSK3 β and coupled signal transduction pathway may play an important role to promote IEC-6 cells apoptosis. Inhibition of GSK3 β activation can provide protective effects against the irradiation induced IEC-6 cells apoptosis. Pretreatment with caspase-3 specific inhibitor has similar effects on IEC-6 cells, being characterized by decreasing apoptotic cell percentage and blockade of DNA fragmentation induced by the irradiation. GSK3 β and coupled signal transduction pathway plays a very important role in the irradiation induced IEC-6 cells injuries. Antagonism of GSK3β and caspase-3 activation may be an important approach to protect intestinal epithelial function. These results are of clinical relevance in antagonism of the irradiation induced intestinal dysfunctions.