Intestinal villi are a unique structural and functional unit for the luminal sensing, digestion, absorption, secretion and immune defense in the small intestine. Subepithelial fibroblasts of the intestinal villi, which form a contractile network beneath the epithelium, are in close contact with epithelial cells, neurons, capillaries, smooth muscles and immune cells, and play pivotal roles in the villous functions. Here, we investigate the functional roles of subepithelial fibroblasts physiologically and morphologically using a primary culture and isolated epithelium-free villi of duodenum isolated from rats, which were anesthetized by pentobarbital (0.1ml/100g) and decapitated. Villous subepithelial fibroblasts possess purinergic receptor P2Y1 and tachykinin receptor NK1. ATP and substance-P (SP) induce increase in intracellular Ca2+ and cell contraction in cultured cells and also isolated villi observed using a two-photon laser microscope with indo-1 calcium dye. The localization of NK1R and SP in the villi was examined by light and electron microscopic immunohistochemistry. NK1R-like immunoreactivity was intensely localized on the plasma membrane of villous subepithelial fibroblasts. The villous subepithelial fibroblasts form synapse-like structures with both SP-immunopositive and -immunonegative nerve varicosities, mostly intrinsic afferents nerve terminals. They are highly mechano-sensitive and stretch stimulation of the cells cultured on an elastic (PDMS) chamber, induced ATP release, which observed by real time luciferin-luciferase bioluminescence imaging system using an image intensifier and a high sensitive EM-CCD camera equipped on an upright microscope. Large transient ATP releases (peak concentration over 10 μM) were observed in sparse cells and the released ATP spreads to the surrounding over several hundred μm and keeps enough concentration to activate P2Y1 for several ten seconds, which forms propagating Ca2+ waves and contraction of the cells. From these findings, we propose that the mutual interaction between villous subepithelial fibroblasts and afferent neurons via SP and ATP plays important roles in refined and coordinate villous movement in intestine.