Homeobox-containing protein 1 (HMBOX1) is a novel member of the homeobox family, and abnormal expression of HMBOX1 has been observed in several types of carcinoma. A total of 144 cases of confirmed glioma diagnoses were included in the present study. Grading was performed according to the World Health Organization (WHO) grading system for central nervous system neoplasm. Immunohistochemical staining of HMBOX1, proliferation marker protein Ki‑67 (Ki‑67) and microvessel density (MVD) was performed, and scores were calculated. HMBOX1 mRNA levels were detected using the reverse transcription quantitative polymerase chain reaction. It was identified that the expression of HMBOX1 was reduced in glioma tissue compared with normal brain tissue (P<0.05). The expression of HMBOX1 was downregulated significantly in WHO grade IV tumors compared with WHO grades II and III (P<0.05). HMBOX1 expression was significantly correlated with WHO grade, Karnofsky Performance Score, MVD and Ki‑67 expression; however, not associated with age or gender. Log‑rank testing did not demonstrate that HMBOX1 expression was associated with prognosis. In conclusion, HMBOX1 may be a potential diagnostic marker in glioma.