Epithelial ovarian cancers (EOC) present as malignant tumors with high mortality in the female reproductive system diseases. Acquired resistance to paclitaxel (PTX), one of the first-line treatment of EOC, remains a therapeutic challenge. ClC-3, a member of the voltage-gated Cl^- channels, plays an essential role in a variety of cellular activities, including chemotherapeutic resistance. Here, we demonstrated that the protein expression and channel function of ClC-3 was upregulated in PTX resistance A2780/PTX cells compared with its parental A2780 cells. The silence of ClC-3 expression by siRNA in A2780/PTX cells partly recovered the PTX sensitivity through restored the G2/M arrest and resumed the chloride channel blocked. ClC-3 siRNA both inhibited the expression of ClC-3 and β-tubulin, whereas the β-tubulin siRNA reduced the expression of itself only, without affecting the expression of ClC-3. Moreover, treatment of ClC-3 siRNA in A2780/PTX cells increased the polymerization ratio of β-tubulin, and the possibility of proteins interaction between ClC-3 and β-tubulin was existing. Take together, the over-expression of ClC-3 protein in PTX-resistance ovarian cancer cells promotes the combination of ClC-3 and β-tubulin, which in turn increase the ration of free form and decrease the quota of the polymeric form of β-tubulin, and finally reduce the sensitivity to PTX. Our findings elucidated a novel function of ClC-3 in regulating PTX resistance and ClC-3 could serve as a potential target to overcome the PTX resistance ovarian cancer.