Forkhead box O3 (FoxO3a), a transcription factor in forkhead family, is associated with the regulation of cell proliferation, metabolism, differentiation and apoptosis. However, whether FoxO3a is involved in the regulation of glucocorticoids induced-hypothalamic-pituitary-adrenal (HPA) dysfunction is still unknown. Our current results showed that dexamethasone(DEX) could upregulate the expression of FoxO3a in PC12 and hypothalamic neuronal cultures while downregulate the expression level of neuropeptide W (NPW), and this process could be blocked by GR2 antagonist. Furthermore, DEX inhibited the phosphorylation of Akt and FoxO3a, but did not inhibit the phosphorylation of ERK1/2, resulting with FoxO3a nuclear localization. We also found that the overexpression of FoxO3a could inhibit NPW expression, while the knockdown FoxO3a by siRNA had the opposite effect, as the regulatory region of NPW promoter contains multiple FoxO3a binding sites, the FoxO3a inhibited NPW’s transcriptional activity by bonding to these sites. In addition, in the rat model, long-term use of corticosteroids could reduce animals’ body weight and sucrose consumption and cause stress- depression like behavior. And corticosterone treatment caused a significant increase in FoxO3a levels, while the expression of NPW protein decreased in the hypothalamus. Meanwhile, FoxO3a and NPW were co-located in the hypothalamus proved by using immunofluorescent double labeling. In summary, these results indicate that NPW is a new direct downstream target gene of FoxO3a. FoxO3a inhibited the transcription of NPW and regulated glucocorticoids-induced HPA dysfunction by directly regulating the expression of NPW. Therefore, the current findings suggest that FoxO3a and NPW may be potential therapeutic targets for endocrine and mental disorders.