ObjectiveTo compare four different transfection reagents for transfection efficiency of rat heart myoblast cells H9C2, to choose the optimal transfection method.MethodsThe plasmids of enhanced green fluorescent protein (EGFP) gene were transfected as exogenous genes to H9C2 cells from four different transfection regents including FuGENE HD, DNA-In CRISPR, Lipofectamine 3000 and Lipofectamine 2000. Fluorescence intensity was measured by fluorescence microscopy and fluorescence microplate reader to evaluate transfection efficiency. The effects of four transfection reagents on cell viability were measured by Cell Counting Kit-8 (CCK-8) reagents.ResultsTransfection efficiency of Lipofectamine 3000 was the highest (>50%), while that of DNA-In CRISPR was the lowest (<1%). The cytotoxicity of Lipofectamine 3000 was the lowest in the four transfection reagents and the cell viability was 94.55% after 48-hour transfection.ConclusionTransfection regent Lipofectamine 3000 has the relatively high transfection efficiency as well as the lowest cytotoxicity, which is more suitable for use in H9C2 cells by transfection.