<i>Purpose:</i> To analyze the prognostic value of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) in patients with locally advanced rectal cancer treated with preoperative radiotherapy. <i>Methods:</i> Eighty-one patients with locally advanced rectal cancer were studied. All patients received preoperative pelvic radiotherapy. Forty-seven patients received concomitant chemotherapy. Surgical resection was performed 4–8 weeks later in all patients. Immunohistochemical examination of COX-2 and VEGF was performed on the preirradiation diagnostic biopsies. An immunohistochemical score established from the extension and intensity of the markers was used for analysis. The log-rank test and proportional hazards regression analysis were used to calculate the probability that the biomarkers were associated with patient outcome. <i>Results:</i> COX-2 expression was positive in 38 tumors (51%) while VEGF expression was positive in 43 (57%). The only clinicopathological parameter significantly associated with COX-2 or VEGF expression was performance status. None of the 2 markers were found to predict treatment response. There was no statistically significant correlation between COX-2 and VEGF. Univariate analysis identified pathological stage (pT, pN) as prognostic for disease-free survival. When VEGF expression was analyzed, disease-free survival was reduced among patients with VEGF-positive tumors (p = 0.047). This was specifically related to metastases-free survival (p = 0.016). These results were not observed for COX-2. After multivariate analysis, the pT and pN stage remained as independent prognostic factors. <i>Conclusions:</i> VEGF-positive expression is an indicator of poor disease-free survival, specifically linked to distant metastasis. More aggressive treatment strategies are warranted in pT3–4 and pN1–2 rectal cancer patients.