Background and Aims Nuclear transcription factor homeobox containing protein 1 (HMBOX1) has different expression patterns in different tumors. And it is closely related to the prognosis of glioma, ovarian cancer, gastric cancer and hepatocellular carcinoma. However, the expression of HMBOX1 and its prognostic significance in colorectal cancer have not been study. Therefore, this study was conducted to investigate the expression of HMBOX1 in colorectal cancer and its relationship with prognosis.Methods The cancer tissue specimens and clinical data of 90 patients who underwent colorectal cancer resection from January 2012 to January 2014 were collected. The expression of HMBOX1 in colorectal cancer tissue was detected by immunohistochemical staining. According to the results of immunohistochemistry, the tissues were divided into high HMBOX1 expression group and low HMBOX1 expression group. The differences in clinicopathologic characteristics and prognosis were analyzed, and the risk factors influencing the postoperative tumor-free survival rate and overall survival rate of colorectal cancer patients were also determined.Results There were 54 (60.0%) cases in high HMBOX1 expression group and 36 (40.0%) cases in low HMBOX1 expression group. The high expression of HMBOX1 was significantly associated with TNM stage, N classification, M classification and histological differentiation (all P<0.05), but irrelevant to age, sex and T classification (all P>0.05). The 1-, 3- and 5-year disease-free survival rates and 1-, 3- and 5-year overall survival rates in high HMBOX1 expression group were all significantly lower than those in low HMBOX1 expression group (all P<0.05). Univariate analysis showed that III-IV stage, N₂, M₁ and high HMBOX1 expression were influencing factors for tumor-free survival rate, and multivariate analysis revealed that III-IV stage, M₁ and high HMBOX1 expression were independent risk factors for tumor-free survival (all P<0.05). Univariate analysis showed that III-IV stage, M₁, poor differentiation and high HMBOX1 expression were influencing factors for overall survival, and multivariate analysis revealed that III-IV stage, low differentiation and high HMBOX1 expression were independent risk factors for overall survival (all P<0.05).Conclusion In colorectal cancer, the expression of HMBOX1 is closely related to the malignant biological properties. It can be used as an index for postoperative prognostic prediction of colorectal cancer patients, and those with high HMBOX1 expression may face a unfavorable prognosis.