It has been suggested that excessive activities or focal segmental glomerulosclerosis (FSGS)-associated gain-of-function mutations of the canonical transient receptor potential 6 (TRPC6) channel may result in a proteinuria and progressive kidney failure. In this study, we investigated the impact of podocin, a mechanosensor at the slit diaphragm of glomerulus, on these enhanced channel activities by Ca²⁺ imaging and patch clamp techniques. Co-expression of podocin suppressed mechanically-enhanced responses of receptor-activated wild-type and 131T-FSGS mutant of TRPC6 channels overexpressed in HEK293 cells. In differentiated cultured podocytes (MPCs) stably overexpressing TRPC6, its mechanical potentiation after receptor activation was found to be decreased as compared with that observed in the heterologous system. However, this decrease was reversed by siRNA knockdown of endogenous podocin expression. These results suggest that the mechanosensitivity of receptor-activated TRPC6 channel may be negatively regulated by interaction with podocin, the mechanism being presumably important to normalize otherwise exaggerated TRPC6-mediated Ca²⁺ responses.