Several genes, including fibroblast growth factor 2 (FGF2), are up-regulated in the hypertrophic heart. However, the molecular mechanisms responsible for the angiotensin II (Ang II)-induced activation of FGF2 in cardiomyocyte hypertrophy are largely unknown. The purpose of this study was to determine the signaling cascades underlying the Ang II-induced transcriptional activation of FGF2 in neonatal rat cardiomyocytes. Real-time quantitative RT-PCR and Western blot showed that Ang II upregulates FGF2 expression and that these effects were attenuated by U0126 or SB203580, but not by SP600125. Deletion analyses revealed that the region between -845 and -666 is essential for Ang II-induced FGF2 promoter activity. The existence of an atypical GATA4-binding motif, located at position -752, was identified using electrophoretic mobility shift assay (EMSA). Using both EMSA and chromatin immunoprecipitation (ChIP) analyses, we also showed that Ang II increases binding of GATA4 to DNA, and that this effect is attenuated in the presence of U0126 or SB203580, but not in the presence of SP600125. GATA4 siRNA significantly reduced Ang II-induced FGF2 mRNA levels. Together, these results indicate that binding of GATA4 to DNA is increased by Ang II via extracellular signal-regulated protein kinase 1/2 (ERK 1/2) and p38 kinase, which increases FGF2 gene expression in neonatal rat cardiomyocytes.