The insulin receptor substrate (IRS) family plays important roles in cellular growth, signaling, and survival in the brain. We identified IRS6/Dok-5, a member of the IRS family, also expressed in heart. Dok-5 expression level significantly increased during cardiomyocyte differentiation of P19CL6 cells. To understand the mechanism of Dok-5 gene expression and regulation during cardiomyocyte differentiation, we first mapped the transcription start site of the mouse Dok-5 gene and characterized its promoter regions. Truncation and mutation analysis of the Dok-5 promoter identified the forkhead binding element responsible for the repression of Dok-5 promoter activation. The co-localization of FOXO3a and Dok-5 in the mouse heart allows FOXO3a to be a transcriptional regulator of Dok-5. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed that FOXO3a could bind to the Dok-5 promoter, accompanied by FOXO3a translocation from the nucleus to cytoplasm. FOXO3a overexpression could inhibit Dok-5 promoter activity. Silencing FOXO3a expression by siRNA upregulated the expression of Dok-5 and enhanced cardiomyocyte differentiation. Moreover, Dok-5 siRNA attenuated cardiomyocyte differentiation. Our results provide the first evidence that FOXO3a, the PI3K/PKB downstream substrate, acts as a transcriptional repressor to inhibit the expression of Dok-5. Dok-5 is involved in cardiomyocyte differentiation by a PI3K/PKB/FOXO3a signaling pathway. Crown Copyright © 2009.