In vascular smooth muscles, the activity of Ca²⁺-activated Cl^- (Cl_Ca) channels regulates the membrane excitability and myogenic tone. TMEM16A channels are predominantly form Cl_Ca currents in vascular smooth muscles including portal vein smooth muscles (PVSMs). Caveola is a cholesterol-rich membrane invaginations and structurally contributes to effective and efficient signal transduction. Caveolin 1 (Cav1) is accumulated in the caveolin and plays a key role in forming the functional complex among enzymes, receptors, and ion channels. In this study, the functional roles of Cav1 on the expression and activity of TMEM16A Cl_Ca channels were examined in portal vein smooth muscle cells (PVSMCs) from wild-type (WT) and Cav1-knockout (KO) mice. Spontaneous contractions of PVSMs were recorded using an isotonic transducer. TMEM16A-mediated Cl_Ca currents were recorded by whole-cell patch-clamp configurations. The expression of TMEM16A channels was quantitatively analyzed by real-time PCR. The amplitude of spontaneous contractions of PVSMs was larger in Cav1-KO mice than WT mice. Whole-cell Cl_Ca currents were also larger in Cav1-KO PVSMCs than WT PVSMCs. Importantly, Ani9 (a specific blocker for TMEM16A channels)-sensitive currents were increased in Cav1-KO PVSMCs compared to WT PVSMCs. The expression of TMEM16A channels was higher in Cav1-KO PVSMs than WT PVSMs. The present data strongly suggest that the caveola structure formed by Cav1 negatively regulates the expression and activity of TMEM16A-mediated Cl_Ca channels in vascular smooth muscle cells.