Differential activation of the Rho family GTPases, Cdc42, Rac1, and RhoA, helps to govern the distinct morphological and migratory phenotypes downstream of adhesion to different extracellular matrix (ECM) proteins. However, it is not known how specific GTPase-dependent signaling pathways are activated in response to different ECM ligands. We hypothesized that adhesion to different ECM molecules, such as collagen and fibronectin, will trigger selective regulation of guanine nucleotide exchange factors (GEFs) to regulate the appropriate matrix-specific cell migratory response. We utilized an affinity precipitation-based mass spectrometry screen to isolate active GEFs from primary human fibroblasts migrating in collagen, fibronectin, or ECM-free environments. Among the GEFs identified, we found that βPix, a Rac1/Cdc42 GEF, was robustly activated only during migration in collagen matrices. Knockdown of βPix led to a collagen-specific migration defect characterized by rapid, spatially-deregulated protrusions, rounded morphology, the absence of stable leading and trailing edges, and robust contraction of the adjacent collagen matrix. In contrast to fibroblasts migrating on fibronectin, βPix in cells migrating in collagen did not localize to focal adhesions, but instead transiently accumulated on the membrane adjacent to areas of cellular protrusion as determined by live cell imaging, immunofluorescence staining, and biochemical iii fractionation. Mechanistically, we found that βPix is critical for efficient migration in fibrillar collagen environments by restraining RhoA signaling. Live FRET imaging and RNAi knockdown established this suppression occurs through a mechanism of Rho GTPase crosstalk between Cdc42 and RhoA that is regulated by a collagen-specific functional interaction between βPix and the GTPase activating protein (GAP) srGAP1. Additionally, we identified that binding of α2β1 integrin to fibrillar collagen leads, through PP2A, to loss of phosphorylation at T526 on βPix and promotes association with srGAP1. We conclude that ECM-dependent regulation of a specific GEF is a fundamental mechanism of migration in different microenvironments. Our results reveal a conserved, matrix-specific pathway controlling migration involving a GEF/GAP interaction of βPix with srGAP1 that is critical for maintaining suppressive crosstalk between Cdc42 and RhoA during 3D collagen migration.