Leucine-zipper and sterile-alpha motif kinase (ZAK) is the key intra-cellular mediator protein in cardiomyocyte hypertrophy induction by transforming growth factor beta 1 (TGF-beta1) which has also been identified as a profibrotic cytokine involved in cardiac fibrosis progression. We hypothesized whether ZAK over-expression causes cardiac scar formation due to the extra-cellular matrix (ECM) degraded enzyme regulation in this paper. Using immuno-histochemical analysis of the human cardiovascular tissue array, we found a positively significant association between ZAK over-expression and myocardial scars. ZAK over-expression in H9c2 cardiomyoblast cells increases the metalloproteinase tissue inhibitor 1/2 (TIMP-1/2) protein level, which reduces matria metalloproteinase-9 (MMP-9) activity and also activates c-JNK N-terminal kinase 1/2 (JNK1/2) and p38 signaling, which induces MMP-2, possibly resulting in cardiac fibrosis. Taken together, ZAK activity inhibition may be a good strategy to prevent the cardiac fibrosis progression.