The present study aimed to identify whether second mitochondria-derived activator of caspase (Smac), vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (Ki-67) expression in pre-treatment tumor biopsies are useful predictive markers of tumor response in patients with rectal cancer undergoing pre-operative chemoradiotherapy (CRT). Paraffin-embedded tissues obtained before and after therapy were evaluated by immunohistochemical staining for Smac, VEGF and Ki-67. The study evaluated the correlation of Smac, VEGF and Ki-67 immunoreactivity in tumor biopsies before treatment of tumor response to pre-operative CRT. Regarding Smac, patients with a favorable response to neoadjuvant CRT had higher pre-therapy levels (p=0.011). The level of Smac expression decreased after neoadjuvant therapy (p=0.044). However, VEGF expression was found to be negatively and significantly correlated with a favorable tumor response to neoadjuvant CRT (p=0.010). A transient increase in VEGF expression was detected in the resected specimens following neoadjuvant therapy (p=0.030). In addition, tumors with a low Ki-67 labeling index (Ki-67-LI) expression were found to be more sensitive to neoadjuvant therapy than those with a high expression of Ki-67-LI (p=0.034). In contrast to VEGF, the Ki-67 expression level decreased after neoadjuvant therapy. Smac, VEGF and Ki-67 expression levels, assessed immunohistochemically from pre-treatment tumor biopsies, may be useful predictive markers of rectal tumor response to pre-operative CRT.