The loss of a DNA mismatch repair occurs in approximately 15% of sporadic colorectal cancer (CRC) and is usually caused by the lack of expression of the hMLH1 gene due to promoter methylation. Despite undergoing adjuvant 5-fluorouracil (5-FU) therapy after a curative surgical resection, some patients with advanced-stage CRC develop recurrence. In the present study, we investigated whether the hMLH1 mRNA expression or promoter methylation is a prognostic factor in CRC patients treated with adjuvant 5-FU. The hMLH1 mRNA expression levels were measured by quantitative reverse transcription PCR in cancer and normal epithelial cells that were obtained from 94 CRC patients using a laser capture microdissection. Then, the methylation status of the hMLH1 promoter in the CRC tissues was examined by methylation-specific PCR. The hMLH1 mRNA expression levels were significantly lower in the cancer cells than in the normal mucosa (p<0.01) and the hMLH1 mRNA expression levels in the cancer cells were significantly lower in the CRC tissues with methylated versus unmethylated hMLH1 (p<0.01) in the 94 patients. Among the 35 patients receiving adjuvant 5-FU, the disease-free survival rate was significantly better in the patients demonstrating a low hMLH1 mRNA expression in the cancer cells in comparison to that of the patients with a high hMLH1 mRNA expression (p<0.01). Moreover, a multivariate analysis revealed that hMLH1 mRNA expression was a significant independent prognostic factor for tumor recurrence in CRC patients treated with adjuvant 5-FU. However, hMLH1 methylation was not correlated with the survival in these 35 patients. These data suggest that the hMLH1 mRNA quantitation in colorectal cancer cells may be helpful for evaluating the prognosis of CRC patients receiving 5-FU-based adjuvant chemotherapy after a surgical resection.