Hepatocellular carcinoma (HCC) is one of the most aggressive malignant diseases and is highly resistant to conventional chemotherapy. Therefore, HCC requires more effective prevention and treatment strategies. 5-fluorouracil (5-FU) remains the most widely used chemotherapeutic drug for the treatment of gastrointestinal, breast, head and neck, and ovarian cancers. In pursuit of a novel effective strategy, we have evaluated the potential of 5-FU to promote endoplasmic reticulum (ER) stress and autophagy in Sk-Hep1 HCC cells. We found that 5-FU profoundly induces ER stress in Sk-Hep1 cells and upregulates p53 and activates CHOP/GADD153 and caspase-12. Activation of CHOP/GADD153 and caspase-12 promotes mitochondrial cell death in Sk-Hep1 cells followed by ER stress. Changes in calcium homeostasis and the protein folding machinery cause stress in the ER, leading to apoptotic cell death. Stress in the ER activates autophagy to remove the misfolded protein aggregates and recover from the stress environment. Our study demonstrates that 5-FU-induced ER stress suppresses autophagy and also downregulates GRP78 expression. Activation of autophagy followed by ER stress facilitates the cell survival response. Therefore, the inhibition of protective autophagy may provide a useful pharmacological target. Taken together, these results indicate that 5-FU-induced ER stress activates the mitochondrial apoptotic cell death pathway by downregulating GRP78 and protective autophagy proteins in Sk-Hep1 cells, raising the possibility of using 5-FU as a therapeutic agent to target human HCC.