Recent evidence suggests that extracellular matrix components may play a signaling role in embryonic valve development. We have previously identified the spatiotemporal expression patterns of periostin in developing valves, but its function during this process is largely unknown. To evaluate the functional role periostin plays during valvulogenesis, two separate three-dimensional culture assay systems, which model chick atrioventricular cushion development, were employed. These assays demonstrated that cushion mesenchymal cells adhered and spread on purified periostin in a dose-responsive manner, similar to collagen I and fibronectin via α_vβ₃ and β₁ integrin pairs. Periostin overexpression resulted in enhanced mesenchyme invasion through 3D collagen gels and increased matrix compaction. This invasion was dependent on α_vβ₃ more than β₁ integrin signaling, and was mediated differentially by Rho kinase and PI 3-kinase. Both matrix invasion and compaction were associated with a colocalization of periostin and β₁ integrin expression to migratory cell phenotype in both surface and deep cells. The Rho/PI 3-kinase pathway also differentially mediated matrix compaction. Both Rho and PI 3-kinase were involved in normal cushion mesenchyme matrix compaction, but only PI 3-kinase was required for the enhanced matrix compaction due to periostin. Taken together, these results highlight periostin as a mediator of matrix remodeling by cushion mesenchyme towards a mature valve structure.