During vascular injury, platelets adhere to exposed subendothelial proteins, such as collagen, on the blood vessel walls to trigger clot formation. Although the biochemical signalings of platelet-collagen interactions have been well characterized, little is known about the role microenvironmental biomechanical properties, such as vascular wall stiffness, may have on clot formation. To that end, we investigated how substrates of varying stiffness conjugated with the same concentration of Type I collagen affect platelet adhesion, spreading, and activation. Using collagen-conjugated polyacrylamide (PA) gels of different stiffnesses, we observed that platelets do in fact mechanotransduce the stiffness cues of collagen substrates, manifesting in increased platelet spreading on stiffer substrates. In addition, increasing substrate stiffness also increases phosphatidylserine exposure, a key aspect of platelet activation that initiates coagulation on the platelet surface. Mechanistically, these collagen substrate stiffness effects are mediated by extracellular calcium levels and actomyosin pathways driven by myosin light chain kinase but not Rho-associated protein kinase. Overall, our results improve our understanding of how the mechanics of different tissues and stroma affect clot formation, what role the increased vessel wall stiffness in atherosclerosis may directly have on thrombosis leading to heart attacks and strokes, and how age-related increased vessel wall stiffness affects hemostasis and thrombosis.