Introduction: Cardiac fibrosis is one of the major hallmarks of heart failure (HF) progression. Fibrosis is initiated as a repair mechanism following any kind of cardiac injury including a heart attack, inflammation, hypertension, etc. Briefly, fibroblasts are activated to form myofibroblasts that secrete collagen. Persistent fibroblast activation leads to excess collagen deposition leading to tissue stiffening and impaired contractility and thereby HF. To date, there is no anti-fibrotic therapy used in HF patients. Using bulk and snRNA sequencing data performed on myocardial tissue from HF patients, we identified adipocyte enhancer binding protein (AEBP1) to be significantly upregulated in activated fibroblasts. AEBP1 has been shown to play a crucial role in fibroblast activation following injury in the kidney, liver, and lungs. AEBP1 inhibition resulted in fibrosis attenuation in these organs. However, its role in cardiac fibrosis is not well understood. Hypothesis: We hypothesize that AEBP1 is crucial for cardiac fibroblast activation and AEBP1 inhibition will prevent collagen secretion and lead to improved cardiac function. Methods: Human cardiac fibroblasts (HCF) from non-failing donors were used for in vitro experiments. AdV-CMV-AEBP1 was used for AEBP1 overexpression (OE) and AdV-CMV-shAEBP1 was used to knockdown (KD) AEBP1 in vitro. AAV9-CMV-shAEBP1 was used for in vivo KD of AEBP1 in mice following myocardial infarction (MI). Human myocardial slices from n=6 HF patients were used and AdV-CMV-shAEBP1 was used to knockdown AEBP1 in human samples. Results and Discussion: AEBP1 OE in HCF resulted in fibroblast activation and collagen secretion (n=6, p=0.002). Contrarily, loss of AEBP1 significantly prevented fibroblast activation, evident from a reduction in transgelin (SM22) (n=6, p=0.02) and collagen (COL1A1) protein levels (n=6, p=0.01). AEBP1 KD in a mouse model of acute cardiac fibrosis resulted in reduced fibrosis and improved cardiac function (n=6, 12% absolute improvement compared to untreated mice, relative improvement in ejection fraction, p=0.02). Tissue culture studies of human HF myocardium showed a significant improvement in cardiac structure evident from reduced cardiomyocyte hypertrophy (n=6 HF patients, p=0.05) and reduced fibrosis (n=6, p<0.0001) following AEBP1 KD. Overall, we show that AEBP1 plays a critical role in fibrogenesis, and its inhibition has the potential to attenuate fibrosis in acute and chronic HF.