2-Aminophenoxazine-3-one (Phx-3) induces apoptosis in several types of cancer cell lines. However, the mechanism of apoptosis induction by Phx-3 has not been fully elucidated. In this study, we investigated the anticancer effects of Phx-3 in the glioblastoma cell line LN229 and analyzed its molecular mechanism. The results indicated that 6- and 20-h treatment with Phx-3 significantly induced apoptosis in LN229 cells, with downregulation of survivin and XIAP. Both ERK and JNK, which are the members of the MAPK family, were activated after treatment with Phx-3. Inhibition of ERK using the specific inhibitor U0126 blocked the Phx-3-induced apoptosis only in part. However, inhibition of JNK using the specific inhibitor SP600125 completely prevented Phx-3-induced apoptosis and restored the phosphorylation states of ERK to the control levels. Enhanced generation of reactive oxygen species (ROS) was detected after 3-h treatment with Phx-3. In addition, the ROS scavenger melatonin almost completely blocked Phx-3-induced JNK activation and apoptosis. This suggests that JNK activation was mediated by Phx-3-induced ROS generation. Although SP600125 and melatonin completely blocked the reduction of mitochondrial membrane potential after a 3-h treatment with Phx-3, extension of Phx-3 exposure time to 20 h resulted in no cancelation of mitochondrial depolarization by these reagents. These reagents also had little effect on the decreased expression of survivin and XIAP during a 3-20-h exposure to Phx-3. These results indicate that the production of ROS following JNK activation is the main axis of Phx-3-induced apoptosis in LN229 cells for short-term exposure to Phx-3, whereas alternative mechanism(s) appear to be involved in apoptosis induction during long-term exposure to Phx-3.