The aim of this study was to explore the regulatory mechanism of retinoic acid (RA) on the TBX1 gene expression in myocardial cells. Ventricular cardiocytes were isolated from neonatal rats and cultured, and then treated with different concentrations of retinoic acid. The expression of Shh and Fgf8 at mRNA and protein levels in neonatal rat myocardial cells were measured by using RT-PCR and Western blot technique, respectively. There was basal expression of Shh and Fgf8 in the control group. When treated with 3×10 mol/L RA, we observed that the expression of Shh mRNA and protein in neonatal rat myocardial cells were up-regulated by 1.51 ( <0.05) and 1.10 times ( <0.05), respectively. In comparison with the control group, under the concentration of 5×10 mol/L RA, they were up-regulated by 2.21 ( <0.05) and 2.38 times ( <0.05) individually. Meanwhile, we could detect that the expression of Fgf8 mRNA and protein were up-regulated by 2.50 times ( <0.05) and 80% ( <0.05) separately compared with the control group after stimulation of 3×10 mol/L RA, and they were up-regulated by 3.48 ( <0.05) and 2.04 times ( <0.05) individually after stimulation of 5×10 mol/L RA. The results indicated that RA could induce the expression of Shh and Fgf8 in neonatal rat myocardial cells. At the same time, it has shown that Shh and Fgf8 were involved in the regulation process of RA on TBX1 expression.