Background and PurposeThe discovery that flavonoids are capable of inhibiting platelet function has led to their investigation as potential antithrombotic agents. However, despite the range of studies on the antiplatelet properties of flavonoids, little is known about the mechanisms by which flavonoids inhibit platelet function. In this study, we aimed to explore the pharmacological effects of a polymethoxy flavonoid, nobiletin, in the modulation of platelet function.Experimental ApproachThe ability of nobiletin to modulate platelet function was explored by using a range ofin vitroandin vivoexperimental approaches. Aggregation, dense granule secretion and spreading assays were performed using washed platelets. Fibrinogen binding, α‐granule secretion and calcium mobilization assays were performed using platelet‐rich plasma and whole blood was used in impedance aggregometry and thrombus formation experiments. The effect of nobiletinin vivowas assessed by measuring tail bleeding time usingC57BL/6 mice.Key ResultsNobiletin was shown to suppress a range of well‐established activatory mechanisms, including platelet aggregation, granule secretion, integrin modulation, calcium mobilization and thrombus formation. Nobiletin extended bleeding time in mice and reduced the phosphorylation ofPKB(Akt) andPLCγ2 within the collagen receptor (glycoproteinVI)‐stimulated pathway, in addition to increasing the levels of cGMPand phosphorylation of vasodilator‐stimulated phosphoprotein, a protein whose activity is associated with inhibitory cyclic nucleotide signalling.Conclusions and ImplicationsThis study provides insight into the underlying molecular mechanisms through which nobiletin modulates haemostasis and thrombus formation. Therefore, nobiletin may represent a potential antithrombotic agent of dietary origins.