Abstract: Objective To investigate the effect of transgiutaminase2(TG2) on hypoxia-induced apoptosis of osteosarcoma cell line MG-63 and the mechanism of TG2 inhibiting cell apoptosis by suppressing the release of Cytochrome C and regulating the expression and activity of Caspase-3. Methods The hypoxia culture model was established by a hypoxia incubator and divided into four groups, normoxia group, pure hypoxia group, control siRNA hypoxia group and TG2 siRNA hypoxia group. The expression of TG2, Cytochrome C and Caspase-3 as well as the apoptosis rate were observed at different hypoxia culture phases (6, 12, 24, 48, 72h). Microtiter plate assay was performed to monitor intracellular TG2 activity. Results Compared with normoxia group, the activity, mRNA level and protein expression of TG2 were increased remarkably with correspondence to the hypoxia time in the pure hypoxia group and control siRNA hypoxia group (P<0.01). But Caspase-3 activity didn't change significantly. Similarly, the protein expression level of Caspase-3 and Cytochrome C in cytosol, as well as the apoptosis rate were increased slightly. In the TG2 siRNA hypoxia group, Caspase-3 activity, Cytochrome C protein expression and cell apoptosis rate were increased obviously (P<0.01). Conclusion In the hypoxia condition, TG2 could inhibit the hypoxia-induced apoptosis through preventing Cytochrome C releasing into the cytosol and suppressing Caspase-3 activities.