Platelets play a very significant role in hemostasis while simultaneously posing a risk for the development of various cardiovascular diseases. Platelet-mediated issues can occur in blood vessels and trigger various medical problems. Therefore, controlling platelet function is important in the prevention of thrombosis. In this regard, we need to find compounds that provide potent antiplatelet activity with minimum side effects. Therefore, we examined the effect of 5-hydroxyindolin-2-one isolated from Protaetia brevitarsis larvae having antiplatelet properties and investigated different pathways that mediate the antiplatelet activity. We examined the effect of 5-hydroxyindolin-2-one (5-HI) on the regulation of phosphoproteins, thromboxane A2 generation, and integrin αIIbβ3 action. Our data showed that human platelet aggregation was inhibited by 5-HI (75, 100, 150, 200 μM) without cytotoxicity, and it suppressed intracellular Ca2+ concentration through the regulation of inositol 1, 4, 5-triphosphate receptor I (Ser1756) and extracellular signal-regulated kinase (ERK). Moreover, collagen-elevated thromboxane A2 production and αIIbβ3 action were inhibited by 5-HI through the regulation of cytosolic phospholipase A2 (cPLA2), mitogen-activated protein kinase p38 (p38MAPK), vasodilator-stimulated phosphoprotein (VASP), phosphoinositide 3-kinase (PI3K), and Akt (protein kinase B). Therefore, we suggested that 5-HI could be a potential substance for the prevention of thrombosis-mediated thrombosis.