The expression levels of hypoxia-inducible factor 1alpha (HIF-1 alpha) and HIF-2 alpha in pancreatic cancer (PC) and their association with clinicopathologic characteristics were investigated in order to elucidate their roles in the development of PC. HIF-1 alpha and HIF-2 alpha mRNA levels in 20 patients with PC were detected by quantitative real-time polymerase chain reaction. The expression of HIF-1 alpha and HIF-2 alpha protein in samples from other 90 patients with PC was measured by immunohistochemistry. Correlations between the expression of HIF-1 alpha or HIF-2 alpha and clinicopathologica features and prognosis were analyzed. The expression of both HIF-1 alpha and HIF-2 alpha mRNA was up-regulated in most cancer tissues (P < 0.05). HIF-1 alpha staining was weakly positive in most cancer tissues and strongly positive in adjacent pancreas tissues (P < 0.05). Clinicopathologic analysis revealed that relatively strong HIF-1 alpha expression in cancer tissues was related to greater invasion (P < 0.05), higher tumor pathologic stage (P < 0.05), higher American Joint Committee on Cancer (AJCC) stage (P < 0.05) and shorter overall survival time (P < 0.05). Conversely, HIF-2 alpha staining was strongly positive in most cancer tissues and weakly positive in adjacent pancreas tissues. Clinicopathologic analysis revealed that relatively strong HIF-2 alpha expression in cancer tissues was related to less invasion (P < 0.05), lower tumor pathologic stage (P < 0.05), lower AJCC stage (P < 0.05) and longer overall survival time (P < 0.05). Moreover, the HIF-1 alpha(high)/HIF-2 alpha(low) group showed a shorter survival time than the HIF-1 alpha(low)/HIF-2 alpha(high) group. In conclusion, although HIF-1 alpha and HIF-2 alpha mRNA expression patterns are the same, their protein expression patterns are significantly different and they play different roles in PC. Combined analysis of HIF-1 alpha and HIF-2 alpha expression might be useful to predict the prognosis of patients with PC.