Interstitial flow emanates from tumors into the microenvironment where it promotes tumor cell invasion. Fibroblasts are key constituents of the tumor stroma that modulate the mechanical environment by matrix remodeling and contraction. Here, we explore how interstitial fluid flow affects fibroblast-tumor cell interactions. Using a 3-dimensional invasion assay and MDA-MB-435S cells cocultured with dermal fibroblasts in a collagen matrix, we showed a synergistic enhancement of tumor cell invasion by fibroblasts in the presence of interstitial flow. Interstitial flow also drove transforming growth factor (TGF)-β1 and collagenase-dependent fibroblast migration, consistent with previously described mechanisms in which flow promotes invasion through autologous chemotaxis and increased motility. Concurrently, migrating fibroblasts enhanced tumor cell invasion by matrix priming via Rho-mediated contraction. We propose a model in which interstitial flow promotes fibroblast migration through increased TGF-β1 activation and collagen degradation, positioning fibroblasts to locally reorganize collagen fibers via Rho-dependent contractility, in turn enhancing tumor cell invasion via mechanotactic cues. This represents a novel mechanism in which interstitial flow causes fibroblast-mediated stromal remodeling that facilitates tumor invasion. Cancer Res; 71(3); 790-800. ©2011 AACR.