Extracellular matrix scaffolds derived from porcine small intestinal submucosa (SIS-ECM) have been shown to promote the formation of site-specific tissue in a number of preclinical animal studies. However, this constructive remodeling process requires that the scaffold be subjected to a site-specific mechanical environment. The specific quantitative effects of mechanical loading on the gene expression patterns of fibroblasts seeded on SIS-ECM are unknown and yet very important in the tissue remodeling process. The objective of the present study was to evaluate the expression of collagen type I (Col I), collagen type III (Col III), smooth muscle actin (SMA), tenascin-C (TN-C), matrix metalloprotease-2 (MMP-2), matrix metalloprotease-9 (MMP-9), transforming growth factor-β1 (TGF-β), and transforming growth factor-β3 (TGF-β3) by fibroblasts subjected to various magnitudes (0%, 5%, 10%, and 15%) and frequencies (0.1 Hz, 0.3 Hz, and 0.5 Hz) of stretch. A new cyclic-stretching tissue culture (CSTC) system was developed. This system consists of eight independently controlled culture chambers that can be operated in a sterile incubator. Each chamber includes a load cell so that the load in each scaffold can be monitored. It was found that different stretching regimens led to complex and distinctive patterns of gene expression by fibroblasts seeded onto SIS-ECM. In general, the fibroblasts increased expression of Col I up to 5-fold and decreased that of Col III with increased frequency of stretch. In addition, the fibroblasts exhibited a contractile phenotype with increased expression of SMA, TN-C, and TGF-β1. These findings support the concept that the mechanical environment of a remodeling ECM scaffold may have substantial effects on the behavior of cells within the scaffold and contribute to the site-specific tissue remodeling that has been observed in in vivo studies.