Background and objective It has been shown that carbonic anhydrase IX (CAIX) was closely related to tumor hypoxia. Proliferating cell nuclear antigen (Ki67) is considered to be an objective indicator which could reliable and comprehensive response to cell population proliferation; vascular endothelial growth factor (VEGF) was positively correlated with tumor angiogenesis. The aim of this study is to investigate the significance of the expression of CAIX in non-small cell lung cancer (NSCLC) tissues by analyzing the correlation between CAIX and clinical characteristics of patients with lung cancer and VEGF, Ki67 expressions. Methods CAIX, VEGF and Ki67 protein were detected in 76 cases of NSCLC and 10 cases of lung inflammatory pseudotumor by immunohistochemistry. Results CAIX was expressed in 45 of 64 cases of NSCLC and not expressed in benign lesion pulmonary tissues. VEGF was expressed in 55 of 76 cases and Ki67 was expressed in 30 of 76 cases. The expression of CAIX in squamous cell carcinoma (SC) was higher than that in adenocarcinoma (AC)(69.7% vs 27.9%, P=0.001). In 34 cases who received radiotherapy, the objective response rates of CAIX positive and negative groups were 27.8% and 62.5% respectively; the expression of CAIX was positively correlated with VEGF (r=0.231, P=0.043) and was not correlated with Ki67 (r=0.064, P=0.583). Conclusion Compared with benign lesion pulmonary tissue, the expression of CAIX in NSCLC tissues is significantly increased and is positively correlated with VEGF. The expression of CAIX was also correlated with the objective response rate of radiotherapy, which provided new evidence about that hypoxia can increase NSCLC radiotherapy resistance.