Chymase is activated after acute myocardial ischemia/reperfusion (AMI-R) and is associated with an early activation of matrix metalloproteinase-9 (MMP-9), which increases infarct size after experimental AMI, and late fibrosis. We assessed the effect of chymase inhibition on myocardial protection and early signs of fibrosis after AMI-R. Fourteen pigs underwent AMI-R and received intravenously either vehicle (V; n ϭ 7) or chymase inhibitor (CM; n ϭ 7). Separately, rat myocardial fibroblast was incubated with vehicle (n ϭ 4), low-dose chymase (n ϭ 4), high-dose chymase (n ϭ 4), or high-dose chymase plus chymase inhibitor (n ϭ 4). Infarct size (V, 41 Ϯ 5; CM, 24 Ϯ 5; P Ͻ 0.01) and serum troponin T (P ϭ 0.03) at the end of reperfusion were significantly reduced in CM. Chymase activity in both the area at risk (AAR) (P ϭ 0.01) and nonischemic area (P ϭ 0.02) was significantly lower in CM. Myocardial levels of pro, cleaved, and cleaved/pro-MMP-9 in the AAR were significantly lower in CM than V (P Ͻ 0.01, Ͻ 0.01, and ϭ 0.02, respectively), whereas phospho-endothelial nitric-oxide synthase (eNOS) (P Ͻ 0.01) and total eNOS (P ϭ 0.03) were significantly higher in CM. Apoptotic cells (P ϭ 0.05), neutrophils (P Ͻ 0.05), and MMP-9-colocalizing mast cells (P Ͻ 0.05) in the AAR were significantly reduced in CM. Interleukin-18 (P Ͻ 0.05) and intercellular adhesion molecule-1 (P Ͻ 0.05) mRNA levels were significantly lower in CM. In cultured cardiac fibrosis, Ki-67-positive cells were significantly higher in the high-dose chymase groups (P Ͻ 0.03). This study demonstrates that chymase inhibition plays crucial roles in myocardial protection related to MMP-9, inflammatory markers, and the eNOS pathway. It may also attenuate fibrosis induced by activated chymase after AMI-R.