Glioblastoma is a malignant primary brain tumor with poor prognosis with a median survival of only 12-15 months. The high mortality rate of this disease is mainly due to the chemoresistance resulting from various reasons. Ubiquitin-specific protease 4 (USP4) has recently been found to be elevated in various types of cancer through regulating P53 activity. However, whether USP4 is responsible for chemoresistance in glioblastoma is not clear. In the present study, the expression of USP4 in glioblastoma tissues and cell lines, as well as its association with temozolomide (TMZ) chemoresistance was analyzed. The results demonstrated that USP4 was significantly upregulated in glioblastoma tissues and cell lines at the mRNA and protein levels. Notably, USP4 knockdown alone did not affect glioblastoma cell viability; however, when USP4 knockdown cells were treated with TMZ, the cell viability was decreased significantly. In addition, the results revealed that cleaved poly(ADP-ribose) polymerase level increased when USP4 was knocked down in glioblastoma cells treated with TMZ. It was also observed that P53 was increased in U251 and U87 cells with USP4 knockdown. Following treatment with a P53 specific inhibitor, the results suggested that USP4 mediated chemoresistance through inhibiting apoptosis in a P53-dependent manner. In conclusion, the data revealed the critical role of USP4 in TMZ resistance in glioblastoma and provided new insight for future drug development for the treatment of this disease.