Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disorder characterized by extensive pulmonary vascular remodeling and right ventricular dysfunction. Recent investigations have identified microRNA-21–5p (miR-21–5p) as a key driver of pulmonary artery smooth muscle cells (PASMCs) phenotypic transformation and extracellular matrix (ECM) dysregulation, thereby exacerbating disease pathology. In this study, we investigated the therapeutic potential of Plumbagin (PL), a natural naphthoquinone compound, in attenuating PAH progression via modulation of the miR-21–5p and ECM remodeling. Using a monocrotaline (MCT)-induced PAH mouse model along with cultured human PASMCs, we evaluated the effects of PL on miR-21–5p expression, bone morphogenetic protein receptor type 2 (BMPR2) levels, and ECM-related factor expression. PL treatment significantly mitigated pulmonary vascular remodeling in the animal model. Mechanistically, PL suppressed miR-21–5p levels, restored BMPR2 expression, and reversed PASMC phenotypic switching, while modulating key ECM regulators including matrix metalloproteinase (MMP)-7, MMP-19, and tissue inhibitor of metalloproteinases-3 (TIMP-3). Clinical validation using serum samples from patients with PAH revealed that elevated miR-21–5p and MMP-7 levels correlated with increased disease severity, whereas higher MMP-19 and TIMP-3 levels were inversely associated. Collectively, these findings highlight targeting the miR-21–5p and ECM dynamics as a promising therapeutic strategy for PAH management and underscore the translational potential of PL in improving patient outcomes.