Reactive oxygen species (ROS) play a profound role in cardiorespiratory function under normal physiological conditions and disease states. ROS can influence neuronal activity by altering various ion channels and transporters. Within the nucleus tractus solitarii (nTS), a vital brainstem area for cardiorespiratory control, hydrogen peroxide (H₂O₂) induces sustained hyperexcitability following an initial depression of neuronal activity. The mechanism(s) associated with the delayed hyperexcitability are unknown. Here we evaluate the effect(s) of H₂O₂ on cytosolic Ca⁺⁺ (via Fura-2 imaging) and voltage-dependent calcium currents in dissociated rat nTS neurons. H₂O₂ perfusion (200 µM; 1 min) induced a delayed, slow, and moderate increase (~27%) in intracellular Ca⁺⁺ ([Ca⁺⁺]_i). The H₂O₂-mediated increase in [Ca⁺⁺]_i prevailed during Thapsigargin, excluding the endoplasmic reticulum as a Ca⁺⁺ source. The effect, however, was abolished by removal of extracellular Ca⁺⁺ or the addition of cadmium to the bath solution, suggesting voltage-gated Ca⁺⁺ channels (VGCCs) as targets for H₂O₂ modulation. Recording of the total voltage-dependent Ca⁺⁺ current confirmed H₂O₂ enhanced Ca⁺⁺ entry. Blocking VGCC L-, N-, and P/Q-subtype decreased the number of cells and their calcium currents that respond to H₂O₂. The number of responder cells to H₂O₂ also decreased in the presence of Dithiothreitol, suggesting the actions of H₂O₂ were dependent on sulfhydryl-oxidation. In summary, here, we have shown that H₂O₂ increases [Ca⁺⁺]_i and their Ca⁺⁺ currents, which is dependent on multiple VGCCs likely by oxidation of sulfhydryl groups. These processes presumably contribute to the previously observed delayed hyperexcitability of nTS neurons in in vitro brainstem slices.