5‐Fluorouracil (5‐FU) is widely used for colorectal cancer (CRC) treatment; however, continuous treatment of CRC cells with 5‐FU can result in acquired resistance, and the underlying mechanism of 5‐FU resistance remains unclear. We previously established an acquired 5‐FU‐resistant CRC cell line, HCT116RF10, and examined its biological features and 5‐FU resistance mechanisms. In this study, we evaluated the 5‐FU sensitivity and cellular respiration dependency of HCT116RF10cells and parental HCT116 cells under conditions of high‐ and low‐glucose concentrations. Both HCT116RF10and parental HCT116 cells were more sensitive to 5‐FU under low‐glucose conditions compared with high‐glucose conditions. Interestingly, HCT116RF10and parental HCT116 cells exhibited altered cellular respiration dependence for glycolysis and mitochondrial respiration under high‐ and low‐glucose conditions. Additionally, HCT116RF10cells showed a markedly decreased ATP production rate compared with HCT116 cells under both high‐ and low‐glucose conditions. Importantly, glucose restriction significantly reduced the ATP production rate for both glycolysis and mitochondrial respiration in HCT116RF10cells compared with HCT116 cells. The ATP production rates in HCT116RF10and HCT116 cells were reduced by approximately 64% and 23%, respectively, under glucose restriction, suggesting that glucose restriction may be effective at enhancing 5‐FU chemotherapy. Overall, these findings shed light on 5‐FU resistance mechanisms, which may lead to improvements in anticancer treatment strategies.