Pulmonary veno-occlusive disease (PVOD) is a rare and severe form of pulmonary arterial hypertension, characterized by progressive obstruction of small pulmonary vessels and a lack of effective therapeutic options. Our previous research, utilizing a mitomycin C (MMC)-induced rat model, demonstrated that activation of the integrated stress response (ISR) via protein kinase R (PKR) is a critical driver of endothelial dysfunction and pulmonary vascular remodeling. However, it remains unclear whether PKR is the sole mediator of ISR activation and the pathogenesis of PVOD under the MMC treatment. In this study, we administered the same dose of MMC used in rats to control (Ctrl) and PKR knockout (KO) mice. Consistent with observations in rats, Ctrl mice exhibited ISR activation in the vascular endothelium and rapidly developed vascular remodeling in both arteries and veins following MMC treatment. In contrast, KO mice showed no evidence of ISR activation or vascular remodeling under same conditions. Proteomic analysis revealed that the PKR-ISR axis perturbs proteostasis in Ctrl mice but not in KO mice. These findings highlight the essential role of PKR-mediated ISR activation and the disruption of proteostasis in the pathogenesis of PVOD, placing PKR as a promising therapeutic target for this disease.