Introduction: Sugen/Hypoxia treated rats and idiopathic pulmonary arterial hypertension (PAH) patients have increased circulating levels of cell-free heme (CFH). However, the mechanisms responsible for the elevated CFH and its pathogenic role are not fully identified. Hypothesis: Mitochondrial energy plays a critical role in the maturation of the red blood cell precursors, reticulocytes (RTC). We propose that incomplete RTC maturation due to mitochondrial dysfunction (MD) could increase RTC fragility and rupture, elevate the plasma CFH and promote PAH. Methods: The rat model of severe MD induced by NFU1 G206C mutation is associated with spontaneous PAH phenotype. This model was used to study erythropoiesis activation, total/immature RTC count, and plasma CFH levels. FITC-dextran extravasation was analyzed in fresh transversely cut pulmonary slices 24h after FITC-dextran injection (60 mg/kg. i.v.) by ChemiDoc MP Imaging System. The effects of free heme in vitro were evaluated using pulmonary arterial smooth muscle cells (PASMCs) isolated from NFU1 G206C and WT rats. Results: NFU1 G206C rats had increased erythropoietin levels and significantly higher total and immature RTC counts compared to WTs. In contrast, NFU1 G206C rats with a restored mitochondrial function, achieved by a chronic supplementation with lipoic acid (LA,1.7 mM orally), had normalized total/immature RTC counts (WT/NFU1 G206C /NFU1 G206C +LA for total and immature RTC: 2.5±0.2%/4.4±0.3%/3.4±0.3%, p<0.002 and 2.2±0.4%/9.8±1.9%/0.8±0.2%, p<0.001, N=8-12). These changes corresponded to the significantly elevated plasma CFH levels and elevated extravasation of FITC-dextral in the lungs in untreated NFU1 G206C rats, suggesting the disruptive effect of CFH on the pulmonary endothelial barrier. Finally, the exposure of PASMC isolated from NFU1 G206C rats to heme (50μM) significantly increased the proliferation rate ~1.5 and ~2.9 folds compared to untreated NFU1 G206C or WT PASMC. Conclusions: We conclude that MD mediates activation of erythropoiesis coupled with an impaired RTC maturation. These effects promote an increase in circulating CFH, disruption of endothelial barrier, and elevated rate of PASMC proliferation, contributing to the PAH pathobiology.