Abstract: Objective To explore the effects of FMS-like tyrosine kinase 3 (Flt3) gene transfection on ischemia reperfusion injury (IRI) in H9c2 rat cardiomyocytes in vitro. Methods A model of IRI was established using H9c2 rat cardiomyocytes. Flt3 gene carried by adenovirus was transfected into H9c2 rat cardiomyocytes. After the model was established, the efficiency of Flt3 gene transfection was detected by immunofluorescence. H9c2 rat cardiomyocytes were divided into four groups: a control group, an IRI group, an Ad-GFP group and an Ad-Flt3 group. The expression of Flt3 and Caspase-3 protein was detected by Western blot. The viability of cardiomyocytes was detected by Cell Counting Kit-8. The levels of lactate dehydrogenase (LDH), creatine kinase MB isozyme (CK-MB), maiondialdehyde (MDA) and superoxide diamutase (SOD) in the supernatant of cell culture were measured by the colorimetric method. Results After IRI, the expression of Flt3 protein decreased, the expression of Caspase-3 protein increased and the viability of cardiomyocytes was significantly up-regulated,compared with the control group(P＜0.05 or P＜0.01). In comparison with non-transfected groups, Flt3 protein expression was enhanced and Caspase-3 protein expression was weakened after transfection of Flt3 gene,compared with the control group (P＜0.05 or P＜0.01).The viability of cardiomyocytes was up-regulated in Flt3 transfected group (P＜0.01). After IRI, the levels of LDH, CK-MB and MDA obviously increased, while the activity of SOD reduced in the supernatant of cell culture,compared with the comtrol group (P＜0.05 or P＜0.01). However, the contents of LDH, CK-MB and MDA were lower in the Flt3 transfected group than those in the non-transfected groups while the activity of SOD was higher (P＜0.05 or P＜0.01). Conclusions Transfection of Flt3 gene can protect cultured H9c2 rat cardiomyocytes against IRI by inhibiting oxidative stress and reducing apoptosis.