Forkhead box O3 (FoxO3a) is a forkhead family transcription factor playing important roles in non-neuronal differentiation, metabolism, proliferation, and survival, but its role in neuronal differentiation remains unclear. In the present study, we investigated the role of FoxO3a in neuronal differentiation and its underlying mechanisms. Our results showed that overexpression of FoxO3a inhibited neuronal differentiation of PC12 cells induced by nerve growth factor (NGF) while knockdown of FoxO3a by siRNA enhanced NGF-induced differentiation. DNA microarray analysis and quantitative reverse transcription PCR (RT-PCR) showed that the overexpression of FoxO3a significantly attenuated expression of neurochondrin (NCDN), a neurite outgrowth-related protein, in PC12 cells, while knocking down the expression of FoxO3a had the opposite effect. Bioinformatic studies found that the regulatory region of NCDN promoter contained multiple FoxO3a binding sites. Dual-luciferase reporter assay with report gene containing NCDN promoter showed that FoxO3a significantly decreased the transcription activity of NCDN promoter. These results indicate that NCDN is a direct downstream target of FoxO3a which negatively regulates the expression of NCDN. Interestingly, NGF-induced NCDN expression and cell differentiation was blocked by the inhibition of phosphatidylinositol-3-kinase (PI3K)-protein kinase B (PKB, Akt) signal pathway (activation of FoxO3a) and overexpression of FoxO3a. Moreover, knockdown of NCDN by siRNA blocked NGF-induced neuronal differentiation of PC12 cells while overexpression of NCDN significantly promoted neurite outgrowth. These results put together demonstrate that NCDN plays an important role in NGF-induced neuronal differentiation and suggest that FoxO3a inhibits NGF-induced neuronal differentiation, at least in part, by suppressing the expression of NCDN.