ClC-3 is a type of chloride channel that has multiple functions in tumorigenesis and tumor growth, and can be blocked by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). In the present study, we found that DIDS inhibited the proliferation of Hep3B hepatocellular carcinoma (HCC) cells in a concentration-dependent manner. More in-depth research demonstrated that DIDS downregulated the protein expression levels of cyclin D1 and cyclin E, which are key proteins of the G1 phase. Additionally, we found that ClC-3 siRNA transfection induced G1 arrest in the Hep3B cells, confirming that ClC-3 is involved in the DIDS-induced inhibition of Hep3B cells. Moreover, the level of α-fetoprotein (AFP), a negative prognostic indicator of HCC, was decreased after treatment with DIDS and ClC-3 siRNA. In conclusion, we demonstrated that ClC-3 can arrest the cell cycle at the G1 phase to inhibit cell proliferation, suggesting that ClC-3 has the potential to be a novel target for HCC therapy and potentially improve the prognosis of HCC patients.