HMGB1 (High Mobility Group Box 1) is a nuclear protein released from damaged cells and implicated in the pathogenesis of pulmonary arterial hypertension (PAH) through activation of proinflammatory and prosurvival responses. However, the role of intracellular HMGB1 signaling, particularly its interactions with DNA and transcriptional regulation, remains underexplored. In this study, we investigated the role of intracellular HMGB1 and its critical residue Cys106 by engineering cell-penetrating peptide (αHMGB1Cys106) that mimics part of the HMGB1 dimeric interface surrounding Cys106. The peptide's effects on HMGB1 intracellular distribution, DNA-binding affinity, and p53 expression and signaling were assessed in cell culture and in vivo using the Sugen/Hypoxia rat model of severe PAH. Our findings demonstrate that αHMGB1Cys106 significantly altered HMGB1 intracellular dynamics, prompting its nuclear exit and subsequent degradation. This effect was associated with a decreased HMGB1-DNA binding and reduced p53 expression both in vitro and in vivo. Remarkably, preventive and therapeutic administration of αHMGB1Cys106 mitigated Sugen/Hypoxia-induced PAH development and progression in a sex-specific manner. In females, the peptide therapy reduced pulmonary apoptosis, senescence, and genotoxic stress, providing significant protection in earlier PAH and effectively reversing the PAH phenotype at advanced stages. Conversely, in males, the treatment yielded only partial benefits, primarily through attenuated genotoxic signaling. These results establish Cys106 as a critical regulator of intracellular HMGB1 signaling, which mediates p53-mediated downstream effects and PAH progression in a sex-specific manner.