The current study examines the association between glucose induction of reactive oxygen species (ROS), mitochondrial (Mt) depolarization, and programmed cell death in primary neurons. In primary dorsal root ganglion (DRG) neurons, 45 mM glucose rapidly induces a peak rise in ROS Corresponding to a 50% increase in mean Mt size at 6 h (P<0.001). This is coupled with loss of regulation of the Mt membrane potential (Mt membrane hyperpolarization, followed by depolarization, MMD), partial depletion of ATP, and activation of caspase‐3 and ‐9. Glucose‐induced activation of ROS, MMD, and caspase‐3 and ‐9 activation is inhibited by myxothiazole and thenoyltrifluoroacetone (P<0.001), which inhibit specific components of the Mt electron transfer chain. Similarly, MMD and caspase‐3 activation are inhibited by 100 fM bongkrekic acid (an inhibitor of the adenosine nucleotide translocase ANT). These results indicate that mild increases in glucose induce ROS and Mt swelling that precedes neuronal apoptosis. Glucotoxicity is blocked by inhibiting ROS induction, MMD, or caspase cleavage by specific inhibitors of electron transfer, or by stabilizing the ANT.—Russell, J. W., Golovoy, D., Vincent, A. M., Mahendru, P., Olzmann, J. A., Mentzer, A., Feldman, E. L. High glucose‐induced oxidative stress and mitochondrial dysfunction in neurons. FASEB J. 16, 1738–1748 (2002)