Objective: To investigate the expression of homeobox gene B4(HOXB4) in glioma tissues and cells, as well as its effects on glioma cell proliferation and invasion, and to explore the upstream regulatory mechanisms of HOXB4 expression. Methods: Based on the Chinese Glioma Genome Atlas (CGGA) database, the expression level of HOXB4 in glioma tissues and its correlation with patient prognosis were analyzed. RT-PCR and Western blot were used to detect HOXB4 expression in U251, U373, and U87 glioma cell lines. The effects of HOXB4 overexpression and silencing on the proliferation and invasion of U87 and U251 cells were assessed using CCK-8 and Transwell assays. U87 cells were treated with inhibitors of AMPK, p38 MAPK, and JNK. The expression of HOXB4 was detected by RT-PCR and Western blot. The cell proliferation and invasion levels were examined by CCK-8 and Transwell assays. HOXB4 was overexpressed in U87 cells in the presence of a JNK inhibitor, and the HOXB4 expression, cell proliferation and invasion were determined by RT-PCR, Western blot, CCK-8, and Transwell assays. Results: HOXB4 was highly expressed in glioma tissues and was associated with tumor malignancy and poor patient prognosis. HOXB4 was expressed in U251, U373, and U87 cell lines, with the highest expression in U87 cells. Overexpression of HOXB4 obviously enhanced the proliferation and invasion of U87 and U251 cells, whereas silencing HOXB4 markedly inhibited these phenotypes. JNK inhibitor significantly downregulated HOXB4 expression in U87 cells, whereas AMPK inhibitor and p38 MAPK inhibitor had no significant effect. JNK inhibitor attenuated the proliferation and invasion of U87 cells, and HOXB4 overexpression antagonized these effects. Conclusion: JNK activation upregulates HOXB4 expression in glioma cells, thereby promoting cell proliferation and invasion.