After injury or cytokine stimulation, fibroblasts transdifferentiate into myofibroblasts, contractile cells that secrete extracellular matrix for wound healing and tissue remodeling. Here, a genome-wide screen identified TRPC6, a Ca²⁺ channel necessary and sufficient for myofibroblast transformation. TRPC6 overexpression fully activated myofibroblast transformation, while fibroblasts lacking Trpc6 were refractory to transforming growth factor β (TGF-β) and angiotensin II-induced transdifferentiation. Trpc6 gene-deleted mice showed impaired dermal and cardiac wound healing after injury. The profibrotic ligands TGF-β and angiotensin II induced TRPC6 expression through p38 mitogen-activated protein kinase (MAPK) serum response factor (SRF) signaling via the TRPC6 promoter. Once induced, TRPC6 activates the Ca²⁺-responsive protein phosphatase calcineurin, which itself induced myofibroblast transdifferentiation. Moreover, inhibition of calcineurin prevented TRPC6-dependent transdifferentiation and dermal wound healing. These results demonstrate an obligate function for TRPC6 and calcineurin in promoting myofibroblast differentiation, suggesting a comprehensive pathway for myofibroblast formation in conjunction with TGF-β, p38 MAPK, and SRF.