Excessive production of reactive oxygen species (ROS) is a critical component of the cellular and molecular pathophysiology of many central nervous system (CNS) disorders, including trauma, ischemia‐reperfusion injury, and neurodegenerative diseases. Hydrogen peroxide (H2O2), an abundant ROS, modulates synaptic transmission and contributes to neuronal damage in the CNS; however, the pathophysiological role of H2O2 in spinal cord ventral horn (VH) neurons remains poorly understood, despite reports that these neurons are highly vulnerable to oxidative stress and ischemia. This was investigated in the present study using a whole‐cell patch clamp approach in rats. We found that exogenous application of H2O2 increased the release of glutamate from excitatory presynaptic terminals and γ‐aminobutyric acid (GABA) from inhibitory presynaptic terminals. The increase of glutamate release was induced in part by an increase in Ca2+ influx through N‐type voltage‐gated calcium channels (VGCCs) as well as by ryanodine receptor (RyR)‐ and inositol triphosphate receptor‐mediated Ca2+ release from the endoplasmic reticulum (ER). In inhibitory presynaptic neurons, increased IP3R‐mediated Ca2+ release from the ER increased GABAergic transmission, which served to rescue VH neurons from excessive release of glutamate from presynaptic terminals. These findings indicate that inhibiting N‐type VGCCs or RyRs may attenuate excitotoxicity resulting from increased glutamatergic activity while preserving the neuroprotective effects of GABA, and may therefore represent a novel and targeted strategy for preventing and treating H2O2‐induced motor neuron disorders. This article is protected by copyright. All rights reserved