--- - |2- Abstract Chloride channel‐3 (ClC‐3), a member of the ClC family of voltage‐gated Cl− channels, is involved in the resistance of tumor cells to chemotherapeutic drugs. Here, we report a new mechanism for ClC‐3 in mediating multidrug resistance (MDR). ClC‐3 was highly expressed in the P‐glycoprotein (P‐gp)‐dependent human lung adenocarcinoma cell line (A549)/paclitaxel (PTX) and the human breast carcinoma cell line (MCF‐7)/doxorubicin (DOX) resistant cells. Changes in the ClC‐3 expression resulted in the development of drug resistance in formerly drug‐sensitive A549 or MCF‐7 cells, and drug sensitivity in formerly drug‐resistant A549/Taxol and MCF‐7/DOX cells. Double transgenic MMTV‐PyMT/CLCN3 mice with spontaneous mammary cancer and ClC‐3 overexpression demonstrated drug resistance to PTX and DOX. ClC‐3 expression upregulated the expression of MDR1 messenger RNA and P‐gp by activating the nuclear factor‐κB (NF‐κB)‐signaling pathway. These data suggest that ClC‐3 expression in cancer cells induces MDR by upregulating NF‐κB‐signaling‐dependent P‐gp expression involving another new mechanism for ClC‐3 in the development of drug resistance of cancers. - Journal of Cellular Physiology, EarlyView.