Dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), has been recently demonstrated as a promising nontoxic antineoplastic agent that promotes apoptosis of cancer cells. In the present study, we aimed to investigate the antitumor effect of DCA combined with 5-Fluorouracil (5-FU) on colorectal cancer (CRC) cells. Four human CRC cell lines were treated with DCA or 5-FU, or a combination of DCA and 5-FU. The cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The interaction between DCA and 5-FU was evaluated by the median effect principle. Immunocytochemistry with bromodeoxyuridine (BrdU) was carried out to determine the proliferation of CRC cells. Cell cycle and apoptosis were measured by flow cytometry, and the expression of apoptosis-related molecules was assessed by western blot. Our results demonstrated that DCA inhibited the viability of CRC cells and had synergistic antiproliferation in combination with 5-FU. Moreover, compared with 5-FU alone, the apoptosis of CRC cells treated with DCA and 5-FU was enhanced and demonstrated with the changes of Bcl-2, Bax, and caspase-3 proteins. Our results suggest that DCA has a synergistic antitumor effect with 5-FU on CRC cell lines in vitro.