Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family of secreted proteins, which include in addition to BDNF, nerve growth factor (NGF) and neurotrophin 3-6 (NT-3-6). BDNF mediates its functions by activating two cell surface receptors, pan-neurotrophin receptor (P75NTR) and tropomyosin-related kinase B (TrkB) and their downstream intracellular cascades. BDNF is best known for its role in neuronal survival, regulation of neuronal differentiation, migration and activity-dependent synaptic plasticity. However, BDNF is widely expressed in non-neuronal tissues as well. The localization and the function of BDNF in intestinal smooth muscle cells (SMCs) are not well defined. Thus, the main purpose of the present study was the identification and characterization of BDNF in intestinal SMCs. Using xviii biochemical and molecular techniques, we have demonstrated in this study that BDNF is synthesized and released in rabbit intestinal longitudinal SMCs cultures. Furthermore, gut neuropeptides, Pituitary Adenylate Cyclase Activating Peptide (PACAP) and substance P (SP) increased BDNF expression and release in SMCs cultures after 24 hrs and 48 hrs incubation. We have also shown that intracellular Ca2+ levels are essential for SP stimulation of BDNF expression and secretion. Lastly, we have demonstrated that exogenous BDNF enhanced carbachol (CCh)-induced contraction of isolated longitudinal muscle strips, and this was inhibited by preincubation with TrkB inhibitor K252a and PLC inhibitor U73122 sugesting that BDNF sensitize longitudinal SMCs to CCh by activating PLC pathway, which is normally absent in those muscle cells. These results provide new insight into the mechanisms of neurotrophin (BDNF) modulation of gut function, which may lead to new therapeutic avenues for treatment of gastrointestinal disorders, and explain some of the pathological changes associated with inflammation such as hypercontractility associated with gut infection or IBD.