Background Evidences show that macrophage migration inhibitory factor(MIF) and the subtype 4 form of glucose transporter(GLUT4) participate in the process of diabetic cardiomyopathy(DCM), however the effect of MIF on modulation of GLUT4 expression is not clear. The present study aimed to investigate the mechanism underlying the modulation of GLUT4 by MIF in cardiomyocytes. Methods The neonatal C57BL/6 mouse ventricular cardiomyocytes(NMVCs) were isolated for in vitro studies. Expressions of GLUT4 and the candidate GLUT4 regulation associated transcription factors in mouse myocardium and NMVCs were determined by quantitative reverse transcription PCR(q RT-PCR), and Western blotting, respectively. The effects of screened transcription factors on mediating MIF-promoted GLUT4 expression were verified by RNA interference(RNAi) assay. Results MIF was upregulated, but GLUT4 was decreased in the diabetic myocardium of the db/db mice. MIF could enhance glucose uptake of the cultured neonatal mouse cardiomyocytes with significant upregulation of GLUT4. RT-q PCR and Western-blot assays showed that MEF2 A,-2C,-2D and Zac1 were significantly up-regulated in MIF-treated NMVCs. Knockdown of Zac1,MEF2 A,-2C, and-2D could markedly inhibit glucose uptake and GLUT4 expression in cardiomyocytes.Moreover, Zac1 was also shown directly regulated by MEF2 A,-2C, and-2D, respectively. Conclusions Transcription factors Zac1 and MEF2 mediate MIF-promoted GLUT4 expression in cardiomyocytes. Background Evidences show that macrophage migration inhibitory factor (MIF) and the subtype 4 form of glucose transporter (MIT) participate in the process of diabetic cardiomyopathy (DCM), however the effect of MIF on modulation of GLUT4 expression is not clear. Methods to investigate the mechanism underlying the modulation of GLUT4 by MIF in cardiomyocytes. Methods The neonatal C57BL / 6 mouse ventricular cardiomyocytes (NMVCs) were isolated for in vitro studies. Expressions of GLUT4 and the candidate GLUT4 regulation associated transcription factors in mouse myocardium and NMVCs were determined by quantitative reverse transcription PCR (q RT-PCR), and Western blotting, respectively. The effects of screened transcription factors on mediating MIF-promoted GLUT4 expression were verified by RNA interference (RNAi) assay. Results MIF was upregulated, but GLUT4 was decreased in the diabetic myocardium of the db / db mice. MIF could enhance glucose uptake of the cultured neonatal mo use cardiomyocytes with significant upregulation of GLUT4. RT-q PCR and Western-blot analysis showed that MEF2 A, -2C, -2D and Zac1 were significantly up-regulated in MIF-treated NMVCs. Knockdown of Zac1, MEF2 A, and-2D could marked inhibit glucose uptake and GLUT4 expression in cardiomyocytes. Moreover, Zac1 also shown directly regulated by MEF2 A, -2C, and-2D, respectively. Conclusions Transcription factors Zac1 and MEF2 mediate MIF- promoted GLUT4 expression in cardiomyocytes.