Objective: To investigate the expressions of the hypoxia marker carbonic anhydrases-IX (CA-IX) in the relevant tissues of the patients with rectal cancer and its effect on the growth of rectal cancer cells in vitro, as well as its role in the tumorigenesis and metastasis of rectal cancer. Methods: The expressions of CA-IX in normal rectal tissues, rectal adenoma, original tumor tissues of rectal cancer, mesorectums and liver metastases were examined by Western blot analysis. The effect of carbonic anhydrases inhibitor acetazolamide on the proliferation of rectal cancer cells in vitro was measured by MTT assay. All the results were analyzed to assess the clinical significance of CA-IX as a tumor marker or metastasis indicator of rectal cancer. Results: The positive rate of CA-IX of the primary tumor tissues of rectal cancer (74.2%) was significantly higher than that of rectal adenoma (16.7%) (P＜0.05), and there was no CA-IX expression in the normal rectal tissues. The expressions of CA-IX in rectal cancer and mesorectal tissues were unrelated to gender, tumor size, differentiation level, clinical stage and liver metastasis of the patients (all P＞0.05). The expression rate of CA-IX in the primary tumor tissues of the N0 patients (5.5%) was lower than that of the N1 (66.7%) and N2 (64.5%) patients (both P＜0.05). The expression rate of CA-IX in mesorectum of the N0 patients (85.5%) was higher than that of N1 (26.2%) and N2 (29.0%) patients (both P＜0.05). Acetazolamide significantly enhanced the ability of 5-fluorouracil of different concentrations to kill the rectal cancer cells in vitro (P＜0.05). Conclusions: CA-IX can accelerate the proliferation of rectal cancer cells and may probably play an important role in the occurrence and development of rectal cancer. It can be used as a tumor marker, but not metastasis indicator of rectal cancer.