Background: TRAF2 and NCK interacting kinase (TNIK) is a serine/threonine kinase member of the germinal center kinase family. Previously, in other cell types, TNIK has been shown to interact with key cytoskeletal regulatory proteins, to regulate F-actin distribution, cell polarization, and neuronal cell arborization. Proteomic studies have demonstrated TNIK is present in platelets; however, no further studies have explored if TNIK plays a role in the regulation of platelet function. Objectives: We sought to investigate the distribution of TNIK in platelets and characterize a potential role for TNIK in platelet function. Methods: The importance of platelet TNIK was explored using 2 structurally distinct TNIK inhibitors (KY-05009 and NCB-0846) in aggregometry, assays of granule secretion, calcium mobilization and thrombus formation, and using confocal microscopy and co-immunoprecipitation. Results: TNIK inhibition diminished platelet responses, and thrombus formation under arterial shear. Furthermore, TNIK regulated platelet adhesion and spreading on fibrinogen and impaired clot retraction due to reduced integrin alpha IIb (3 3 activation. Coimmunoprecipitation studies demonstrated that TNIK bridges interactions between the actin cytoskeleton and integrin alpha IIb (3 3 through direct associations with focal adhesion kinase, paxillin, kindlin-3, filamin A, Rap2a, gelsolin and actin. Conclusion: In summary, we confirm TNIK is present in platelets and is important for platelet function, and thrombus formation. For the first time, we show that platelet TNIK is able to associate with key cytoskeletal regulators, linking its signaling to integrin alpha IIb (3 3 function. Future studies will focus on exploring a potential role of TNIK in the regulation of thrombosis and hemostasis in vivo to assess TNIK's potential as a novel antithrombotic target.