The resistance of colon cancer to 5-fluorouracil (5-FU) is a critical issue, and the cause of this resistance cannot always be explained based on the known molecules. Heat shock protein 27 (HSP27) mRNA expression has recently been shown to be correlated with 5-FU resistance in 5-FU-resistant cell lines. In this study, we attempted to elucidate the functional mechanism of HSP27 in 5-FU resistance in colon cancer. HSP27 protein levels in several human colon cancer cell lines (LoVo, HCT15, WiDr, HCT116, HT-29 and SW480) were determined by immunoblot and densitometry analysis. The in vitro growth inhibition rates (IR) of the cell lines at various concentrations of 5-FU were assessed by MTT assay. The degree of 5-FU resistance was estimated as the drug concentration inducing 50% IR (IC50). The HSP27 protein level and IC50 were significantly correlated in these cell lines (p=0.010). The effect of HSP27 overexpression on IC50 was evaluated in LoVo cells. HSP27 transfectants significantly increased IC50 and reduced HSP27 resistance. The effect of HSP27 down-regulation by HSP27 siRNA on IC50 was confirmed in HCT15 cells. HSP27 siRNA suppressed HSP27 protein levels and reduced IC50 in a dose-dependent manner. These data indicated that HSP27 is closely connected with 5-FU resistance in colon cancer and suggested that HSP27 levels predicted 5-FU resistance. HSP27 down-regulation overcame 5-FU resistance and HSP27 may be a clinical target in patients with 5-FU-resistant colon cancer.