Calmodulin (CaM) is an important modulator of various types of ion channels and Ca²⁺-dependent cellular processes. All mammalian TRPC proteins appear to contain CaM binding sites. TRPC4 possess three C-terminal CaM binding domains. CaM binds to the channel in a Ca²⁺-dependent way. In the present study we have explored the specific roles of the CaM binding sites in the modulation of the channel activity by selectively disrupting each binding site and heterologous expression of the wild type and mutant channels in Hek cells. Using Ca²⁺ imaging, we show that disruption of specific CaM binding domains in TRPC4 abolished Ca²⁺ oscillations in response to stimulation with low doses of carbachol in Hek cells. Furthermore, disruption of all CaM binding sites almost abolished Ca²⁺ mobilization under carbachol stimulation. We also performed patch-clamp experiments to analyze the CaM mediated channel modulation. Activation of G-proteins in cells stably expressing wild type TRPC4 results in activation of outwardly rectifying currents. In contrast, cells expressing the mutated TRPC4 protein lacking all CaM binding sites failed to respond to stimulation. We report that CaM binding to TRPC4 is important for the channel activation.