5-Fluorouracil (5-FU) is widely used for treatment of advanced colorectal cancer. However, it is common for such patients to develop resistance to 5-FU, and this drug resistance becomes a critical problem for chemotherapy. The mechanisms underlying this resistance are largely unknown. To screen for proteins possibly responsible for 5-FU resistance, cells resistant to 5-FU were derived from human colon cancer cell lines and two-dimensional gel electrophoresis–based comparative proteomics was done. Two-dimensional gel electrophoresis data showed there was lower expression of the α subunit of mitochondrial F₁F₀-ATP synthase (ATP synthase) in 5-FU–resistant cells compared with parent cells. Western blotting showed that expression of other ATP synthase complex subunits was also lower in 5-FU–resistant cell lines and that these resistant cells also showed decreased ATP synthase activity and reduced intracellular ATP content. The ATP synthase inhibitor, oligomycin A, strongly antagonized 5-FU–induced suppression of cell proliferation. When 5-FU sensitivity was compared with ATP synthase activity in six different human colon cancer cell lines, a positive correlation has been found. Furthermore, suppressed ATP synthase d-subunit expression by siRNA transfection increased cell viability in the presence of 5-FU. Bioenergetic dysfunction of mitochondria has been reported as a hallmark of many types of cancers (i.e., down-regulation of ATP synthase β-subunit expression in liver, kidney, colon, squamous oesophageal, and lung carcinomas, as well as in breast and gastric adenocarcinomas). Our findings show that ATP synthase down-regulation may not only be a bioenergetic signature of colorectal carcinomas but may also lead to cellular events responsible for 5-FU resistance.