Forkhead box O3 (FoxO3a), one of the FoxO transcription factors, is involved in cell proliferation, metabolism and apoptosis. But the role and mechanism of FoxO3a on neural cell differentiation is not well known. In This study we investigated the role of FoxO3a on neuronal differentiation. In NGF induced PC12 cell differentiation model, we found that overexpression of FoxO3a inhibited NGF-induced neuronal differentiation and the expression of Growth Associated Protein 43 (GAP-43), a neuronal differentiation regulator. Knockdown of FoxO3a expression by specific siRNA had the opposite effect. Bioinformatics studies found that the GaP-43 promoter region contained multiple FoxO3a binding sites. To investigate whether GAP43 is FoxO3a target gene, the GAP-43 promoter were cloned into pGL3-basic and the transcriptional activity were detected with dual luciferase assay. Results showed that Foxo3a significantly decreased transcriptional activity of GAP-43 promoter and the mutation in FoxO3a potential binding sites reversed the inhibition effect. These findings suggest that GAP-43 is a direct downstream target of FoxO3a and its transcription activity is negatively regulated by FoxO3a. Moreover, NGF promoted the expression of GAP-43 mRNA and protein level in a dose- and time-dependent manner. Overexpression of GAP-43 enhanced the differentiation of PC12 cells induced by NGF, suggesting that GAP-43 plays an important role in NGF-induced neural differentiation. Furthermore, NGF promoted the phosphorylation of Akt, ERK and FoxO3a, which induced FoxO3a cytoplasm location. While the Akt and MEK pathway inhibitors blocked the expression of GAP-43 induced by NGF, suggesting that PI3K/Akt and MEK is involved in the action of NGF. In conclusion, our studies showed that FoxO3a inhibits NGF-induced neural differentiation through transcriptionally suppresses GAP-43 expression and Akt and MEK pathways are involved.