Objective To investigate the levels of cystathionine-beta-synthase (CBS) in colon cancer tissues compared with adjacent control tissues; and to examine the relationship between CBS level and clinical characteristics and prognosis. Methods This retrospective study enrolled patients with primary colon cancer. Paraffin-embedded specimens were used to create pathological tissue microarrays. Immunohistochemistry was performed on the microarray to detect the levels of CBS in colon cancer tissues and normal adjacent tissues. Analyses were undertaken to examine the relationship between the level of CBS and clinical characteristics and prognosis. Results A total of 216 patients (107 males and 109 females) were included in the study. The level of CBS in cancer tissues was found to be significantly increased compared with normal adjacent control tissues. There were significant differences in tumour location, tumour-node-metastasis stage and survival rate between the CBS-negative and CBS-positive groups. Positive CBS immunostaining was associated with decreased survival in colon cancer patients. The results of multivariate Cox regression analysis revealed that tumour location and positive CBS immunostaining were independent prognostic factors for survival. Conclusion Positive CBS immunostaining was closely associated with colon cancer and high levels of CBS might accelerate tumour development and affect patient prognosis in colon cancer.