Development of functional engineered matrices for regenerative therapies can benefit from an understanding of how physical cues at the microscale affect cell behavior. In this work, we use microfabricated systems to study how stiffness and microscale topographical cues in the form of “micropegs” affect extracellular matrix synthesis. Previous work from our lab has shown that microtopographical cues in 2D and 3D systems decrease cellular proliferation and regulate matrix synthesis. In this work, the combined role of stiffness and topography on ECM synthesis is investigated in a 2D micropeg system. These studies show that fibroblasts cultured on polydimethylsiloxane (PDMS) substrates with micropegs have reduced expression of collagen type I (Col I) and collagen type VI (Col VI) compared to fibroblasts cultured on flat substrates. In addition, cells on micropegged substrates exhibit down-regulation of other important regulators of ECM synthesis such as α-smooth muscle actin (α-SMA), and integrin α3 (Int α3). Interestingly, this effect is dependent on the contractility and adhesion of the cells. When cultured in the presence of RhoA kinase (ROCK) and myosin light chain kinase (MLCK) inhibitors, no significant differences in the expression of collagen, α-SMA, Int α3, and TGFB1 are observed. Additionally, disruptions in cell adhesion prevent microtopographical regulation of ECM synthesis. When using an antibody to block the extracellular domain of Int α3, no differences in the expression of collagen are observed and blocking Int α3 results in enhanced down-regulation of α-SMA on the stiffer micropegged substrates. These findings demonstrate that regulation of extracellular matrix production by cells on a synthetic substrate can be guided via physical cues at the microscale, and add to the body of knowledge on the role of integrin-mediated mechanotransduction.