Abstract Body (Do not enter title and authors here): Introduction: Estrogen receptor α (ERα) promotes cardioprotective signaling in right ventricle cardiomyocytes (RVCMs). NLRP3 inflammasome activation in macrophages contributes to RV failure (RVF) development in pulmonary hypertension (PH). However, NLRP3 signaling in RVCMs has not been studied. We hypothesized that NLRP3 inflammasome activation mediates RVCM contractile dysfunction and is attenuated by 17β-estradiol (E2) via ERα. Methods: RVs from male or female PH patients with RVF were assessed for NLRP3 activation by analyzing RNA-seq and proteomics data, plus staining for NLRP3 and its binding partner ASC. Male or female human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were treated with endothelin-1 (ET1) ± E2. RVCMs were isolated from male or female monocrotaline (MCT) or pulmonary artery banding (PAB) rats. RVCMs were also isolated from healthy rats and treated with ET1 ± E2 in vitro. Male or female ERα loss-of-function mutant (ERα^mut)] rats were employed to study effects of ERα. NLRP3 activation in RVCMs and iPSC-CMs was assessed by NLRP3-ASC colocalization and activation of downstream targets. RVCM contractility and cytosolic calcium (c-Ca²⁺) were evaluated via IONOPTIX system. P<0.05 was considered significant. Results: RNA-seq, proteomics, and immunofluorescence studies revealed that upregulation of NLRP3 activity and signaling in human RVF are more pronounced in males (p<0.05 for all comparisons). In male patients, NLRP3 activation was inversely correlated with cardiac index (p<0.05). In pilot studies, NLRP3 was more activated in male vs female ET1-treated iPSC-CMs. RVCMs from male, but not female, MCT- or PAB-rats demonstrated increased NLRP3-ASC colocalization (p<0.05). RVCMs treated with ET1 exhibited more NLRP3 activation and contractile dysfunction than female RVCMs (p<0.05). E2 treatment in male WT RVCMs reduced NLRP3-ASC co-localization and increased Ca²⁺ dependent contractility (p<0.05). E2 treatment in male iPSC-CMs trended to prevent ET1-induced NLRP3 activation. E2’s inhibitory effects on NLRP3 activation, NLRP3-induced contractile dysfunction, and c-Ca²⁺ in male WT RVCMs were abrogated in ERα^mut RVCMs (p<0.05). Conclusion: NLRP3 activation in human and rat RVF as well as NLRP3-induced contractile dysfunction in human iPSC-CMs and rat RVCMs exhibit a male bias. E2, via ERα, prevents NLRP3 activation and NLRP3-induced contractile dysfunction. Inhibiting NLRP3 via E2-ERα may be a novel treatment strategy for RVF.