There is an urgent need to search for novel chemosensitizers in the field of cancer therapy. Chlorogenic acid (CGA), a type of polyphenol present in the diet, has many biological activities. The present study is designed to explore the influence of CGA on the effects of 5-fluorouracil (5-FU) on human hepatocellular carcinoma cells (HepG2 and Hep3B). Treatment with 5-FU induced the inhibition of hepatocellular carcinoma cells' proliferation, and the combined treatment with CGA enhanced this inhibition. 5-FU also increased the production of reactive oxygen species (ROS). The combination of 5-FU and CGA led to a more prominent production of ROS and significantly inactivated ERK1/2, although CGA and 5-FU exerted no significant changes when used alone. A previous report has shown that ROS are upstream mediators that inactivate ERK in hepatocellular carcinoma cells. Combined with our results, this indicates that the combination of 5-FU and CGA leads to the inactivation of ERK through the overproduction of ROS. This mediates the enhancement of 5-FU-induced inhibition of hepatocellular carcinoma cells' proliferation, that is, CGA sensitizes hepatocellular carcinoma cells to 5-FU treatment by the suppression of ERK activation through the overproduction of ROS. CGA has shown potential as a chemosensitizer of 5-FU chemotherapy in hepatocellular carcinoma.