D-24851 is a recently developed microtubule inhibitor that induces G2/M cell-cycle arrest and has an antitumor effect in many cancer cell types. It is expected to be a promising chemotherapeutic agent against a broad range of tumors. However, the precise mechanisms underlying its antitumor effect remain to be determined. Here, we investigated the in vitro effect of D-24851 on tumor growth and the apoptosis mechanism in human malignant glioma cells. Because both p53-dependent and -independent pathways of apoptosis have been reported, we used cell lines with wild-type p53 (U87-MG and D54) and cell lines with mutant p53 (U373-MG and T98G) and compared their responses to D-24851. D-24851 substantially inhibited the proliferation of the four glioma cell lines tested in a dose- and time-dependent manner. The inhibitory effect of D-24851 on tumor growth was associated with cell-cycle arrest in G2/M, subsequently inducing apoptosis. D-24851 treatment induced phosphorylated Bcl-2 and translocated Bax from the cytoplasm to the mitochondria, resulting in apoptotic cell death. These events took place regardless of the p53 status of tumor cells. Our results indicated that D-24851 effectively induces apoptosis through Bcl-2 phosphorylation and Bax translocation in human malignant glioma cells in a p53-independent manner. The results of this study make D-24851 even more promising as a therapeutic agent, especially because many malignant gliomas have a heterogeneous p53 status.