This study was focused on the mechanisms of the protective effects of L-arginine and the nitric oxide donor SNAP under the induction of cyclosporine A-dependent mitochondrial permeability transition pore (MPTP) by excess of Ca2+ or D,L-palmitoylcarnitine (PC). The possible involvement of a signaling system that included mitochondrial enzymes NO-synthase (mtNOS), guanylate cyclase (GC), and cGMP-dependent protein kinase G (PKG) (mtNOS/GC/PKG-SS) in the regulation of MPTP was investigated. The experiments were performed on isolated liver mitochondria using inhibitory analysis. The results of the study have shown that L-arginine and SNAP caused a dose-dependent effects on the opening of MPTP. L‑Arginine and SNAP at low concentrations (5–100 μM) activated protective mechanisms, providing an increase in the mitochondrial calcium buffer capacity (CRC) and the critical (threshold) concentration of palmitoylcarnitine (PC*) required for MPTP induction. At higher concentrations, L-arginine (more than 500 μM) and SNAP (more than 100 μM) caused the opposite effect, that is, they suppressed respiration and promoted pore opening. Inhibitors of NOS, GC, and PKG eliminated the protective effects observed at low concentrations of L‑arginine and SNAP. The experiments conducted with a specific inhibitor of inducible NOS (W1400) showed that this calcium-independent enzyme did not participate in the regulation of MPTP in our experiments. Based on the results obtained, it can be assumed that the calcium-dependent mitochondrial signaling system mtNOS/GC/PKG-SS is involved in the regulation of MPTP, providing an increase in CRC and PC*. At high concentrations of L-arginine or SNAP, excess of NO overcame the protection provided by mtPKG and promoted pore opening.