The Rho family of small guanosine triphosphatases (Rho GTPases: RhoA, Cdc42, and Rac1) regulates many aspects of cell behavior, including actin dynamics and cell migration. The generation of calcium ion (Ca²⁺) microdomains is critical in promoting cell migration because they control the localized activity of Rho GTPases. We identified receptor-activated TRPC5 and TRPC6 (transient receptor potential canonical type 5 and 6) channels as antagonistic regulators of actin remodeling and cell motility in fibroblasts and kidney podocytes. We show that TRPC5 is in a molecular complex with Rac1, whereas TRPC6 is in a molecular complex with RhoA. TRPC5-mediated Ca²⁺ influx induces Rac1 activation, thereby promoting cell migration, whereas TRPC6-mediated Ca²⁺ influx increases RhoA activity, thereby inhibiting cell migration. Our data unveil antagonistic Ca²⁺ influx pathways as a conserved signaling mechanism for the integrated regulation of cell migration.