Abstract Body: We previously demonstrated that VSMCs Notch3 regulates brown adipose tissue (BAT) production of adiponectin and anti-contractile effects. Nox4-derived H2O2 is involved in BAT protective effects in the vasculature. Here, we investigated whether Nox4 plays a role in Notch3 regulation of BAT biology and if the Notch3/Nox4 vascular-BAT interactome is altered in hypertension. We characterized Notch3 signaling in BAT from Nox4 KO mice (N4KO). We evaluated expression of Notch3 and its intracellular domain (activation), BAT markers (UCP-1, IL-6), adiponectin and markers of inflammation (CD45, TNFα) and fibrosis (Col1A1, IL-11, galectin-3) by qPCR and WB. Isolated arteries from control mice were exposed to BAT conditioned medium (CM) and vascular function assessed by myography. Expression of Notch3 (N4KO: 0.60±0.01; WT: 1.06±0.02 AU, p<0.05) and its ICD (N4KO: 0.55±0.008; WT: 0.72±0.01 AU, p<0.05) were reduced in Nox4 KO BAT; followed by reduced expression of UCP-1 (N4KO: 0.72±0.04; WT: 1.05±0.06 AU, p<0.05) and gene levels of IL-6 (N4KO: 0.56±0.04; WT: 1.31±0.18 2^-ddCt, p<0.05). We did not observe an increase in adiponectin levels in BAT from Nox4 KO. BAT from Nox4 KO mice demonstrated increased levels of pro-inflammatory/fibrotic markers: CD45 (N4KO: 2.55±0.17; WT: 1.0±0.02 2^-ddCt, p<0.05); IL-11 (N4KO: 1.34±0.06; WT: 1.01±0.08 2^-ddCt, p<0.05) and galectin-3 (N4KO: 7.1±0.19; WT: 1.01±0.06 2^-ddCt, p<0.05). The anti-contractile effect of BAT CM was blunted in Nox4 KO conditions. Similar phenotype was found in BAT from LinA3 mice, an Ang II-dependent experimental model of hypertension, where Notch3/Notch3 ICD expression was not altered, followed by an increase pro-inflammatory/fibrotic markers and no changes in BAT markers expression (p<0.05 vs. WT). Nox4 plays an important role in Notch3 regulation of BAT biology, including production of adiponectin and expression of BAT markers. Blunted Notch3/Nox4 axis in adipose tissue during hypertension may lead to BAT dysfunction, inflammation and fibrosis, impacting vascular function and structure. These novel findings suggest an important role for Notch3 in the vascular:adipose interactome in health and cardiovascular diseases.