Introduction: Altered expression of non-coding RNAs (ncRNAs) has been connected to many cardiac disease conditions (1). A hallmark of these conditions is insufficient oxygen supply (hypoxia). Some studies already showed that ncRNAs are transported by extracellular vesicles (EVs) and transferred to their target cells (2,3). However, if ncRNA-enriched vesicles might be involved in myocyte-fibroblast crosstalk during hypoxia is not known so far. In this study, the paracrine communication mechanisms between hypoxic cardiomyocytes and cardiac fibroblasts via extracellular vesicles and the impact of hypoxic cardiomyocyte-derived vesicles mediating cardiac fibrosis is addressed. Methods and Results: Murine cardiomyocytes (HL-1 cells) were exposed to normoxic (21% O2) and hypoxic (0.2% O2) conditions for 24h following 4h reoxygenation. EVs were purified from the conditioned medium of hypoxic and normoxic cardiomyocytes by differential centrifugation and ultracentrifugation steps. To investigate the morphology of isolated vesicle subsets, including apoptotic bodies, microvesicles and exosomes, we used transmission electron microscopy, showing a typical shape and structure of all vesicle subtypes. EVs were further analyzed by Western Blot and nanoparticle tracking, which revealed that cardiomyocyte-derived vesicles differ in protein composition and concentration. Moreover, we studied the secretion and uptake mechanisms of cardiomyocyte-derived vesicles by confocal laser microscopy and found that the vesicles were taken up by fibroblasts in a temperature- and time-dependent manner. To examine the effect on fibroblasts, we co-cultured them with conditioned medium of normoxic and hypoxic cardiomyocytes and measured the gene expression of fibrotic markers by qPCR. We observed that conditioned medium of hypoxic cardiomyocytes led to an increase in fibrotic markers such as collagen 1, collagen 3, connective tissue growth factor (p<0.05, n=4) and matrix metalloproteinase 2 (p=0.064, n=4) in fibroblasts (P-values are determined by Student’s t-tests). Co-culture of fibroblasts with individual fractions of isolated vesicles revealed that mainly the microvesicle fraction contributes to a pro-fibrotic response. The results further indicate a paracrine cardiomyocyte-fibroblast crosstalk mediated by microvesicles. Moreover, analysis of the RNA content of cardiomyocyte-derived vesicles identified a large amount of small RNAs, but also larger RNAs which might play a role in this paracrine communication. Conclusion: In response to hypoxia, cardiomyocytes produce and secrete different subtypes of EVs which are taken up by fibroblasts, triggering a fibrotic response in cardiac fibroblasts. Moreover, cardiomyocyte-derived vesicles contain small non-coding RNAs which might be involved in the paracrine crosstalk between cardiomyocytes and cardiac fibroblasts during hypoxia.