Cardiac fibroblasts and myofibroblasts are responsible for post-MI remodeling which occurs via regulation of extracellular matrix (ECM). Accelerated post-MI remodeling leads to excessive ECM deposition and fibrosis, contributing to impaired contractile function, arrhythmias, and heart failure. We have previously reported that type VI collagen induces myofibroblast differentiation in cultured cardiac fibroblasts, and that type VI collagen and myofibroblast content were both elevated in the myocardium 20 weeks post-MI. The purpose of this study was to determine the expression patterns of type VI collagen and myofibroblast content in early post-myocardial infarction (MI) remodeling to gain insight into whether type VI collagen induces in vivo myofibroblast differentiation via specific matrix–receptor interactions. Adult male Sprague-Dawley rats were anesthetized and left coronary arteries were permanently ligated. Histological tissue sections and whole tissue protein lysates were obtained from infarcted and non-infarcted areas of MI hearts and sham operated controls. At 3 days post-MI, we observed a significant increase in α₃ integrin expression (2.02±0.18 fold); at 7 days post-infarction both type VI collagen (2.27±0.18 fold) and myofibroblast (4.65±0.6 fold) content increased. By 14 days myofibroblast content returned to sham control levels, although type VI collagen (2.42±0.11 fold) was still elevated. In vitro cross-linking confirmed that the α₃ integrin interacts with type VI collagen, and α₃ integrin function blocking antibodies inhibited the differentiation of isolated cardiac fibroblasts. Collectively, our in vitro results indicate that the α₃ integrin receptor interacts with type VI collagen to promote myofibroblast differentiation, and that this interaction may impact in vivo post-MI remodeling.