Abstract: The cellular mechanisms that underlie the diverse nitrosative stress-mediated cellular events associated with ischemic complications in endothelial cells are not yet clear. To characterize whether autophagic elements are associated with the nitrosative stress that causes endothelial damage after ischemia injury, an in vitro sustained oxygen–glucose deprivation (OGD) and an in vivo microsphere embolism model were used in the present study. Consistent with OGD-induced peroxynitrite formation, a rapid induction of microtubule-associated protein 1 light chain 3 (LC3)-I/II conversion and green fluorescent protein-LC3 puncta accumulation were observed in endothelial cells. The Western blot analyses indicated that OGD induced elevations in lysosome-associated membrane protein 2 and cathepsin B protein levels. Similar results were observed in the microvessel insult model, following occlusion of the microvessels using microsphere injections in rats. Furthermore, cultured endothelial cells treated with peroxynitrite (1–50 μm) exhibited a concentration-dependent change in the pattern of autophagy–lysosome signaling. Intriguingly, OGD-induced autophagy–lysosome processes were attenuated by PEP-19 overexpression and by a small-interfering RNA (siRNA)-mediated knockdown of eNOS. The importance of nitrosative stress in ischemia-induced autophagy–lysosome cascades is further supported by our finding that pharmacological inhibition of nitrosative stress by melatonin partially inhibits the ischemia-induced autophagy–lysosome cascade and the degradation of the tight junction proteins. Taken together, the present results demonstrate that peroxynitrite-mediated nitrosative stress at least partially potentiates autophagy–lysosome signaling during sustained ischemic insult-induced endothelial cell damage.