Nitric oxide (NO) inhibits TRPC3 channels via a PKG-dependent mechanism. We sought to determine 1) if NO inhibition of TRPC3 occurs in freshly isolated smooth muscle cells (SMC) and 2) if NO inhibition of TRPC3 channels contributes to NO-mediated vasorelaxation. We tested these hypotheses in freshly isolated rat carotid artery (CA) SMC using patch clamp and in intact carotid artery by vessel myograph. We demonstrated TRPC3 expression in whole CA (mRNA and protein) that was localized to the smooth muscle layers. TRPC1 protein was also expressed and co-immunoprecipitated with TRPC3. Whole cell patch clamp demonstrated non-selective cation channel currents that were activated by UTP (60 µM) and completely inhibited by a TRPC channel inhibitor, La³⁺ (100 µM). The UTP-stimulated current (I_UTP) was also inhibited by intracellular application of anti-TRPC3 or anti-TRPC1 antibody but not by anti-TRPC6 or anti-TRPC4 control antibodies. We next evaluated the NO signaling pathway on I_UTP. Exogenous NO (MAHMA NONOate) or a cell permeable cGMP analog (8Br-cGMP) significantly inhibited I_UTP. Pre-application of a PKG inhibitor (KT5823) reversed the inhibition of MAHMA NONOate or 8Br-cGMP, demonstrating the critical role of PKG in NO inhibition of TRPC1/TRPC3. Intact CA segments were contracted with UTP (100 µM) in the presence or absence of La³⁺ (100 µM), and then evaluated for relaxation to an NO donor, sodium nitroprusside (1 nM to 1 µM). Relaxation to sodium nitroprusside was significantly reduced in the La³⁺ treatment group. We conclude that freshly isolated SMC express TRPC1/TRPC3 channels and that these channels are inhibited by NO/cGMP/PKG. Furthermore, NO contributes to vasorelaxation by inhibition of La³⁺-sensitive channels consistent with TRPC1/TRPC3.