Previously, we have shown that the selective mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel opener BMS-191095 (BMS) induces neuronal preconditioning (PC); however, the exact mechanism of BMS-induced neuroprotection remains unclear. In this study, we have identified key components of the cascade resulting in delayed neuronal PC with BMS using isolated rat brain mitochondria and primary cultures of rat cortical neurons. BMS depolarized isolated mitochondria without an increase in reactive oxygen species (ROS) generation and induced rapid phosphorylation of Akt and glycogen synthase kinase-3beta. Long-term (3 days) treatment of neurons with BMS resulted in sustained mitochondrial depolarization, decreased basal ROS generation, and elevated ATP levels. This treatment also elicited almost complete protection against glutamate excitotoxicity, which could be abolished using the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin, but not with the superoxide dismutase (SOD) mimetic M40401. Long-term BMS treatment induced a PI3K-dependent increase in the expression and activity of catalase without affecting manganese SOD and copper/zinc-dependent SOD. Finally, the catalase inhibitor 3-aminotriazole dose-dependently antagonized the neuroprotective effect of BMS-induced PC. In summary, BMS depolarizes mitochondria without ROS generation, activates the PI3K-Akt pathway, improves ATP content, and increases catalase expression. These mechanisms appear to play important roles in the neuroprotective effect of BMS.