The present study investigates whether the expression levels of heat shock protein 27 (HSP27) in colon cancer cells are associated with 5-fluorouracil (5-FU) sensitivity in a xenograft model, as well as the mechanism responsible for regulating 5-FU sensitivity. HCT116 cells which have a high expression of HSP27 were stably transfected with specific short hairpin RNA (shRNA) in order to suppress HSP27 expression. The association between HSP27 protein expression levels and 5-FU sensitivity was evaluated in a mouse xenograft model. The mRNA expression of 5-FU metabolic enzymes and cell apoptosis were also analyzed in the transfected cells. The suppression of HSP27 protein expression led to enhanced 5-FU sensitivity. The mRNA expression levels of dihydropyrimidine dehydrogenase and orotate phosphoribosyltransferase, but not those of thymidylate synthase, and the number of apoptotic cells increased in the transfected cells after 5-FU exposure. In conclusion, the suppression of HSP27 expression in colon cancer cells may promote 5-FU sensitivity by inducing apoptosis, despite the acceleration in 5-FU metabolism.