Estrogen plays important roles in regulation of bone formation. Chloride channels in the ClC family are expressed in osteoblasts and are associated with bone physiology and pathology, but the relationship between chloride channels and estrogen is not clear. In this study, the action of estrogen on chloride channels was investigated in osteoblastic MC3T3-E1 cells. Results showed that 17β-estradiol could activate a current which reversed at a potential close to Cl^– equilibrium potential with the anion selectivity I- >Br->Cl->gluconate and was inhibited by the chloride channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and knockdown of ClC-3 chloride channel expression. Extracellular application of membrane-impermeable 17β-estradiol-albumin conjugates activated a similar current. The estrogen-activated chloride current could be inhibited by the estrogen receptor antagonist fulvestrant (ICI 182780). The selective ERα agonist, but not ERβ agonist activated a chloride current similar to that induced by 17β-estradiol. Silencing ERα expression prevented activation of estrogen-induced currents. The G protein-coupled estrogen receptor (GPR30) agonist G-1 could not activate chloride channels and the GPR30 antagonist G-15 failed to inhibit estrogen-activated currents. Immunofluorescence and co-immunoprecipitation experiments demonstrated that ClC-3 chloride channels and ERα were co-localized and closely related in cells. Estrogen promoted translocation of ClC-3 and ERα to the cell membrane from the nucleus. In conclusion, it is founded that chloride channels can be activated by estrogen via the ERα receptors located in the cell membrane and suggests that the ClC-3 chloride channel may be one of the action targets of estrogen in regulation of osteoblastic activities.