Nesfatin-1, a satiety molecule processed from nucleobindin2 (NUCB2), is implicated in visceral hypersensitivity in rats and colocalized with 5-hydroxytryptamine (5-HT) in the dorsal raphe nucleus (DRN). Maternal separation (MS) in rats contributes to visceral hypersensitivity via elevated expression of 5-HT in the DRN. Intracerebroventricular injection of nesfatin-1 activates DRN 5-HT neurons. In this study, A model of visceral hypersensitivity was developed by subjecting rats to MS. Colorectal distension was used to detect visceral sensitivity, which was evaluated by abdominal withdrawal reflex (AWR) scores and electromyogram (EMG) magnitude. MS rats exhibited higher AWR scores and EMG magnitude compared with controls. The numbers of nesfatin-1- and tryptophan hydroxylase (TPH, the rate-limiting enzyme for 5-HT synthesis)-positive cells in the DRN were significantly elevated accordingly. Visceral hypersensitivity was significantly alleviated in MS rats treated with intra-DRN administration of anti-nesfatin-1/NUCB2, accompanied by decreased expression of 5-HT and TPH in the DRN, compared with the vehicle-treated group. In contrast, intra-DRN administration of nesfatin-1 into normal adult rats induced visceral hypersensitivity, which correlated with elevated expression of 5-HT and TPH in the DRN. In conclusion, Nesfatin-1 has critical effects on visceral hypersensitivity; the underlying mechanisms might be related to the activation of DRN 5-HT neurons.