Abstract Body (Do not enter title and authors here): Background: Interleukin-27 (IL-27) promotes atherosclerosis and IL-1β production by activating the NLRP3 inflammasome in macrophages and monocytes, but IL27 also suppresses inflammation by stimulating Tregs’ proliferation, IL10 production, and Th17 differentiation. However, the effect and underlying mechanism of IL27 on cardiac inflammation and HF development is still incompletely understood. Hypothesis and Methods: We hypothesize that IL27 exerts an important role in promoting cardiac inflammation and heart failure development through stimulating both cardiac immune cells and nonimmune cells using both pharmacological and genetic modulating IL27 signaling in mice. Results: We found that Transverse Aortic Constriction (TAC)-induced heart failure was associated with significant increase of plasm and cardiac IL27 in mice. In addition, we found administration of recombinant mouse IL27 significantly exacerbated TAC-induced cardiac accumulation of multiple immune cell subsets, cardiac fibrosis, cardiomyocyte hypertrophy, and heart failure (HF). Conversely, blockade of IL27 signaling by IL27 antibody significantly suppressed TAC-induced cardiac accumulation of multiple immune cell subsets and IFNg⁺ T cells, cardiac fibrosis, cardiomyocyte hypertrophy, HF, and death. In addition, genetic inhibition of IL27 signaling by IL27 receptor A (IL27RA) knockout also significantly suppressed TAC-induced cardiac inflammation, fibrosis, cardiomyocyte hypertrophy, HF, and death in mice. Both IL27RA KO or IL27 antibody attenuated cardiac CD8 T cell activation and accumulation. Furthermore, single cell RNA-seq showed that IL27 receptors are expressed in T cells, cardiomyocytes, and adipocytes. RNA-seq showed that IL27RA knockout drastically suppressed multiple immune signaling pathways associated with immune response, antigen presentation capacity, as well as downregulated signaling pathways related to inflammation, pathogen infection, extracellular matrix formation, degradation and organization. Conclusions: Our findings demonstrates that IL27 plays a critical role in promoting cardiac inflammation and HF development through modulating both cardiac immune cells (mainly T cells) and nonimmune cells (such as cardiomyocytes).