Oxidative stress and mitochondrial dysfunction play critical roles in neurodegenerative diseases. Glutathione (GSH), a key brain antioxidant, helps to neutralize reactive oxygen species (ROS) and maintain redox balance. We investigated the effectiveness of L-cysteine (L-Cys) in preventing apoptosis induced by the ROS generator 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) in mouse hippocampal neuronal HT22 cells, as well as alleviating memory and cognitive impairments caused by the GSH synthesis inhibitor L-buthionine sulfoximine (BSO) in mice. DMNQ-induced apoptotic events in HT22 cells, including elevated cytosolic and mitochondrial ROS levels, DNA fragmentation, endoplasmic reticulum stress, and mitochondrial damage-mediated apoptotic pathways were dose-dependently abrogated by L-Cys (0.5–2 mM). The reduced intracellular GSH level, caused by DMNQ treatment, was restored by L-Cys cotreatment. Although L-Cys did not significantly restore GSH in the presence of BSO, it prevented DMNQ-induced ROS elevation, mitochondrial damage, and apoptosis. Furthermore, compared to N-acetylcysteine and GSH, L-Cys had higher 2,2-diphenyl-1-picrylhydrazyl and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid radical-scavenging activity. L-Cys also restored mitochondrial respiration capacity in DMNQ-treated HT22 cells by reversing mitochondrial fission–fusion dynamic balance. BSO administration (500 mg/kg/day) in mice led to neuronal deficits, including memory and cognitive impairments, which were effectively mitigated by oral L-Cys (15 or 30 mg/kg/day). L-Cys also reduced BSO-induced ROS levels in the mice hippocampus and cortex. These findings suggest that even though it does not contribute to intracellular GSH synthesis, exogenous L-Cys protects neuronal cells against oxidative stress-induced mitochondrial damage and apoptosis, by acting as a ROS scavenger, which is beneficial in ameliorating neurocognitive deficits caused by oxidative stress.