Objective To investigate the feasibility of transfecting bone marrow mesenchymal stem cells (BMSCs) with Nkx2.5 to enhance cardiomyogenic differentiation. Methods Rat BMSCs were isolated by the whole bone marrow culture and amplified by serial subcultivation. CD90 and CD45 of the third passage cells were identified by flow cytometry. Using the cationic liposome reagent, Lipofectamine 2000, the plasmid pEGFP-N1-Nkx2.5 was transfected into BMSCs. The transfected cells were observed under an inverted fluorescence microscope and expression of Nkx2.5 was examined with immunocytochemistry. Expression of cardiac troponin T(cTnT) and GATA4 were determined with Western blotting and immunocytochemistry. Results 99% of the third passage cells were positive for CD90 and negative for CD45 on flow cytometry. After 48 hours of transfection, some cells in the transfected group expressed the green Nkx2.5-EGFP fusion protein and Nkx2.5 was expressed only in pEGFP-N1-Nkx2.5 transfected group (n =3). After 4 weeks, Western blotting results indicated that the expression of cTnT in pEGFP-N1-Nkx2.5 transfected group was significantly higher than those in pEGFP-N1 vector transfected group and in control one (EM>n/EM> =3). Immunocytochemistry results showed that the expressions of cTnT and GATA4 in pEGFP-N1-Nkx2.5 transfected group were respectively the highest in the three groups ( EM>n/EM> =3). Conclusion Exogenous expression of Nkx2.5 gene may enhance the cardiomyogenic differentiation of BMSCs.