Second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) is released by mitochondria in response to apoptotic stimuli and is thought to regulate apoptosis by antagonizing inhibitors of apoptosis proteins, which play an important role in sensitization of cancer cells to various therapeutic regimens. The expression of Smac/DIABLO has been demonstrated in various cancer cells, though little is known about its clinical significance with respect to colorectal cancer. The current study was designed to evaluate the relationship between prognosis and Smac/DIABLO expression by clinicopathological analysis of patients with colorectal cancer. Smac/DIABLO expression was evaluated using immunohistochemical staining in 121 consecutive patients with colorectal cancer and the relationship between Smac/DIABLO expression and clinicopathological factors was analyzed. Smac/DIABLO-positive expression was detected in 80 of the 121 patients (66%). The incidence of lymph node and distant metastasis in Smac/DIABLO-negative cancer was significantly higher than that in Smac/DIABLO-positive cancer (P=0.0004 and P=0.003, respectively). While univariate analysis showed that survival in patients with Smac/DIABLO-negative expression was significantly poorer than in Smac/DIABLO-positive cases (P<0.0001), Smac/DIABLO-negative expression was a prognostic indicator independent of Dukes' staging and lymph node metastasis by multivariate analysis. This study proposes that the decrease of Smac/DIABLO expression is an independent factor determining the poorer prognosis of patients with colorectal cancer.