Brain-derived neurotrophic factor (BDNF) may play a critical role in gut motility. We aimed to investigate BDNF's physiologic effects on gut motility in slow-transit constipation (STC) and to explore the underlying molecular mechanisms. BDNF expression and alterations of colonic nerve fibre density in STC patients were first investigated. BDNF's effects on gastrointestinal motility of both BDNF(+/-) mice and loperamide-induced constipation mice were then examined in vivo and in vitro. Smooth muscle α-actin (α-SMA) expression, and nerve fibre, neuromuscular junction (NMJ) and smooth muscle cell (SMC) alterations were investigated. Finally, the effects of BDNF-induced TrkB-phospholipase C/inositol trisphosphate (TrkB-PLC/IP3) pathway activation on gut motility were investigated. In STC patients, BDNF expression and nerve fibre density were decreased, and mucosal nerve fibre ultrastructural degenerations were demonstrated. Gut motility was decreased in vivo and in vitro in BDNF(+/-) and constipation mice, with BDNF dose-dependently increasing gut motility. In BDNF(+/-) mice, α-SMA expression and nerve fibre density were decreased, and nerve fibre, NMJ and SMC ultrastructural degenerations were observed. Finally, TrkB-PLC/IP3 pathway antagonists dramatically attenuated BDNF's excitatory effect on gut motility, and exogenous BDNF induced an obvious increase in IP3 expression. BDNF plays an important regulatory role in gut motility in STC. It was mediated by altering the intestinal innervation structure, as well as smooth muscle secondary degeneration through a mechanism involving TrkB-PLC/IP3 pathway activation.