Abstract Naïve macrophages (Mϕ) polarize in response to various environmental cues to a spectrum of M1 or M2 macrophages with distinct biological functions. Our earlier in vivo studies demonstrated that Notch1 deficiency attenuates the development of abdominal aortic aneurysm by promoting TGF-β2 dependent M2-polarization, however, the underlying mechanism is not completely understood. The present studies aimed to determine the specific roles for Notch1 signaling in macrophage polarization. Bone marrow derived macrophages isolated from WT or Notch1+/− mice (n=12) were differentiated to Mϕ, M1 or M2-phenotypes by 24h exposure to vehicle, LPS/IFN-γ or IL4/IL13, respectively. Total RNA isolated from these cells was subjected to RNA-Sequencing in triplicate. All samples had a minimum of 51.3 million 50 nucleotide paired-end reads and aligned at 87.5% ± 0.5% to the reference genome. EasyRNAseq and WebGestalt analysis with log2 fold change demonstrated that Notch1 haploinsufficiency downregulated the expression of 262 genes at basal level, 307 genes with LPS/IFN-γ and 254 genes with IL4/IL13 treatment. Among these, 50 unique genes including fibromodulin (Fmod), caspase-4, Has1, Col1a1, Alpl and Igf were downregulated by Notch1 haploinsufficiency under all the conditions. Pathway analysis demonstrated that extracellular matrix, macrophage polarization and osteogenesis to be the major pathways affected by Notch1 haploinsufficiency. Gain and loss-of-function and mechanistic studies established a strong correlation between Notch1 haploinsufficiency and Fmod in regulating TGF-β signaling. Overall, our studies provide strong evidence that Notch1 haploinsufficiency increases M2 polarization through these newly identified genes.