Purpose: Increasing attention has recently been directed to the pivotal role of myofibroblasts in the pathogenesis of fibrogenic stenosis and inflammation. Available evidence suggests that part of myofibroblastic function may reflect altered Ca2+ homeostasis. We thus explored a potential role of canonical transient receptor potential (TRPC) channels expressed therein by using human colonic myofibroblast cell line InMyoFib. Here we show that transforming growth factor (TGF)-β1, the major stimulator of intestinal fibrosis, requires TRPC4 and TRPC6-mediated Ca2+ influx for its effect on myofibroblast differentiation and interleukin synthesis. Methods: InMyoFib were grown in SmBM medium supplemented with 5%FBS and growth factors (insulin, hFGF-B, hEGF, FBS and gentamicin/ amphotericin-B). Immunobotting and immunoprecipitation were performed to detect the expression and interactions of proteins, and Ca2+ influx through TRP channels was evaluated by digital imaging of Fura-2 fluorescence. The results were compared between control and TGF-β1(5ng/ml in 1%FBS/SmBM)-treated groups of InMyoFib and statistically evaluated by single and multiple comparison tests. Results: the expressions of fibrosis marker α-smooth muscle actin (α-SMA) and cadherin were detected with TRPC6 and TRPC4, respectively. The expression level of α-SMA and cadherin was augmented by TGF-β1 stimulation, and this was significantly suppressed by treatment with SK&F96365 or transfection of TRPC4- and TRPC6-specific siRNA or their dominant negatives, respectively. Biochemical analysis indicated that the calcineurin-NFAT signaling is involved in the effects. In realtime-PCR experiments, the production of four interleukins from InMyoFib was found significantly accelerated by siRNA knockdown and dominant negative transfection of TRPC6. Detailed immunoblotting and biochemical analyses suggested that Ca2+ influx through TRPC4 and TRPC6 may prevent the phosphorylation of Smad2, Erk1/2, and p38 MAPK proteins at the downstream of TGF-β receptors. Conclusion: from these results, it is speculated that TRPC4 and C6 channels may play non-trivial roles in myofibroblast transformation/differentiation via NFAT signaling, thereby promoting fibrogenesis in the colon. It is also suggested that myfibroblast signaling pathways regulating interleukin production, which seem associated with the phosphorylation of Smad2, Erk1/2, and p38 MAPK as well as TRPC6-mediated Ca2+ influx, could be a promising target in treating fibrotic and inflammatory bowel diseases.