Vascular endothelial growth factor (VEGF) is required for the development, growth and survival of blood vessels. Endothelial cell behavior is altered by cell substrate stiffness, suggesting that VEGF activity might also be influenced by cell-substrate mechanics. We studied VEGF binding, internalization, and signaling as a function of substrate stiffness using endothelial cells cultured on fibronectin (fn) linked polyacrylamide gels. Individual cell analysis of VEGF-induced calcium fluxes in endothelial cells on various stiffness extracellular matrices (ECM) revealed heterogeneity in our cell population that would have been lost using population based averaging. Cluster analysis of individual cells identified two key groups of reacting cells- a minor fraction of highly reactive cells and the bulk of the cells with minimal activation. At subsaturating VEGF doses, highly active cells were phenotypically smaller and thinner than the bulk population. Overall, cells on our softest substrates (4 kPa) were most sensitive to VEGF. To better understand the mechanisms underlying the changes in VEGF signaling due to stiffness, we explored how matrix binding of VEGF and tethering of cells to the matrix modulates VEGF processing. VEGF-ECM binding was enhanced with heparin pre-treatment, which exposed a cryptic VEGF binding site in the fn ECM. Cell produced ECM on the softest substrates were least responsive to heparin, but the cells internalized more VEGF and showed enhanced VEGF signaling compared to cells on all other substrates. Inhibiting VEGF-matrix binding with sucrose octasulfate decreased cell-internalization of VEGF in all conditions. β1 integrin, which connects cells to fn, modulated VEGF uptake in a stiffness dependent fashion. β1 protein levels were consistent with stiffness, yet cells on hard surfaces showed greater decreases in VEGF internalization than cells on softer matrices after β1 inhibition. Stiff matrices facilitate the unfolding of fn, which may reduce the binding capacity of β1 integrin. Thus a greater proportion of activated β1 integrin may be sensitive to inhibition in the stiff condition as compared to the soft. Ultimately, through analysis of individual and population-based VEGF-cell responses to stiffness, this study provides insight into how signaling dynamics, cell heterogeneity, and microenvironment influence tissue regeneration and response to injury and disease.