The Cl^– secretion via Ca²⁺-activated Cl^– channel (CaCC) is critical for fluid secretion in exocrine glands like the salivary gland. Also in the mammary gland, it has been hypothesized that CaCC plays an important role in the secretion of Cl^– and aqueous phase of milk. However, there has been no evidence for the functional expression of CaCC in native mammary secretory (MS) cells of lactating animals. We therefore assessed the membrane current in the MS cells that were freshly isolated from lactating mice using the whole-cell patch-clamp techniques. In the MS cells, we detected the CaCC current that exhibited the following characters: 1) Ca²⁺-dependnet activation at the concentrations of submicromolar range, 2) voltage-dependent activation, 3) slow kinetics for activation and deactivation, 4) outward rectification of the steady-state current, 5) anion permeability in the sequence of I^– > NO₃^– > Br^– > Cl^– >> glutamate, 6) inhibition by Cl^– channel blockers (NFA, DIDS, and CaCCinh-A01). These characters of the native CaCC current were similar to reported characters of heterologously expressed TMEM16A. RT-PCR analyses showed the expression of multiple CaCC channels including TMEM16A, Best1, and Best3 in the mammary glands of lactating mice. Immunohistochemical staining revealed the localization of TMEM16A protein at the apical membrane of the MS cells. Collectively, our data strongly suggest that MS cells functionally express CaCC, which is at least partly constituted by TMEM16A. The CaCC such as TMEM16A at the apical membrane of the MS cells may influence the quantity and/or quality of milk.