Abstract Body: Excess extracellular matrix (ECM) production by activated cardiac fibroblasts (CFs) induces cardiac fibrosis and heart failure. Reactive oxygen species (ROS) production in both CFs and myocytes drives cellular dysfunction, myocyte cell death, and worsened fibrosis. Prohibitin 1 (PHB1) is a critical regulator of mitochondrial integrity that is increased in activated CFs. We posit that PHB1 protects activated CF mitochondrial function, thus preventing ROS-induced cell death. We hypothesize that PHB1 deletion will reduce the fibrogenic capacity of activated CFs. To investigate the impact of CF-specific Phb1 deletion in vivo, we crossed Postn-driven inducible Cre mice (Postn^MCM) with Phb1^fl/fl mice. Adult males and females (13–16 weeks) were infused with Isoproterenol (ISO, 10 mg/kg/day) for one week via osmotic micro-pumps and were compared to age-matched untreated mice. All mice were injected with tamoxifen (75 mg/kg, i.p.) on days 1-3 of ISO to produce activated CFs deficient in Phb1 (Postn-Phb1^-/-) and Phb1 replete controls (Postn-Phb1^+/+). Fibrosis was quantified in mid-myocardial sections via Sirius Red staining, and protein expression was determined by western blot. ISO induced significant increases in cardiac hypertrophy, periostin levels, collagen 1 gene expression, and fibrosis in both sexes. However, males demonstrated more robust increases in collagen gene expression and fibrosis compared to females (sex x ISO interaction). Overall, females had significantly less fibrosis than males, and Postn-Phb1^-/- females tended to have less fibrosis than Postn-Phb1^+/+ females (p=0.07). Further, Phb1 deletion attenuated Postn expression in both sexes implying a pro-fibrogenic role for CF PHB1. ISO increased NOX2 (p91^phox and p67^phox) in male and female Postn-Phb1^+/+ mice. Fibrosis and p91^phox were positively correlated in Postn-Phb1^+/+ males only. This correlation along with the induction p91^phox were prevented by Phb1 deletion in males, suggesting that increased fibrosis in males depends on a signaling axis between CF PHB1 and cardiac NOX2 that may not be present in females. In conclusion, CF PHB1 mediates fibrosis in both sexes, but there are sex-specific impacts on cardiac ROS signaling that may differentially impact ECM remodeling.