Rationale: S100 proteins released from necrotic cells function as danger signals, referred to as alarmins, which play an active role in tissue inflammation and repair via recognition of molecular pattern receptors such as Toll-like receptors (TLRs). Objective: To investigate whether S100A1, the S100 isoform with the highest abundance in cardiomyocytes, acts as an alarmin in response to ischemic myocardial injury. Methods and Results: Patients with acute ST-elevation myocardial infarction (MI) revealed a significant increase in circulating S100A1 protein serum levels assessed by enzyme-linked immunosorbent assay. Experimental MI in C57Bl/6 mice induced comparable S100A1 release, and immunofluorescence uncovered S100A1 internalization in cardiac fibroblasts (CFs) adjacent to damaged cardiomyocytes in vivo . Further in vitro analyses using genetic, biochemical and imaging techniques demonstrated exclusive and rapid endocytosis of S100A1 by CFs, followed by endosomal TLR4-dependent transient activation of mitogen- and stress-activated protein kinases (MAPK/SAPK) and nuclear factor kappaB component p65. CFs exposed to S100A1 assumed an anti-fibrotic and immunomodulatory phenotype exemplified by decreased collagen and smooth muscle actin and enhanced intercellular adhesion molecule 1 and interleukin 10 mRNA and protein levels downstream of MAPK/SAPK and p65. In C57Bl/6 mice, intramyocardial S100A1 injection recapitulated the specific transcriptional changes in vivo . Subsequently, antibody-mediated neutralization of MI-released S100A1 enlarged infarct size and enhanced pro-fibrotic marker expression. Conclusion: Our study identifies cardiomyocyte damage-released S100A1 as cardiac alarmin and uncovers the molecular mechanisms conveying anti-fibrotic and immunomodulatory actions. The beneficial impact of the S100A1-mediated TLR4-dependent cardiac fibroblast phenotype conversion warrants continued investigation of its role in myocardial infarct healing.