We tested the hypotheses that the NO-cGMP-PKG pathway mediates inhibition of the store-operated cation channel (SOC) in human glomerular mesangial cells (HMC) and that TRPC4, a molecular component of SOC in HMC, is associated with PKG-phosphorylated vasodilator-stimulated phosphoprotein (VASP). Using fura 2 ratiometry, we measured intracellular Ca²⁺ concentration [Ca²⁺]i to determine whether sodium nitroprusside (SNP), an NO donor, and 8-Br-cGMP affected SOC-TRPC4 via PKG. We found that the SOC response in HMC was attenuated in the presence of 100 µM SNP, an NO donor, or 100 µM 8-Br-cGMP. Addition of DT-3 (2.5 µM), a specific protein kinase G-1 (PKG-1) inhibitor, reversed the effects of 8-Br-cGMP on the SOC response. Application of 100 µM db-cAMP did not significantly inhibit the SOC response. RT-PCR and western blot revealed transcript and protein in HMC. Immunocytochemical analysis localized PKG-1 to the cytoplasm and plasma membrane of HMC. Previous studies have shown that PKG-mediated phosphorylation of VASP attenuates cellular Ca²⁺ entry resulting in altered growth and proliferation. Therefore, we used western blot and immunocytochemistry to determine whether PKG-phosphorylated VASP associates with TRPC4. Western blot analysis revealed that 8-Br-cGMP enhanced the phosphorylation of VASP at serine 239 (Ser239), a known PKG phosphorylation site, in HMC within 5 minutes. Co-IP and co-immunostaining showed that P-Ser239-VASP associated with TRPC4. However, VASP that was unphosphorylated at Ser239 was not associated with TRPC4. These results indicate that VASP has a role in the NO/PKG-1 mediated inhibition of the TRPC4-SOC response in HMC.