Protein tyrosine phosphatase non-receptor type 22 (PTPN22) is a protein tyrosine phosphatase and negatively regulates T cell signaling. However, whether it is expressed and functions in platelets remains unknown. Here we investigated the expression and role of PTPN22 in platelet function. We showed PTPN22 expression in both human and mouse platelets. Using PTPN22-/- mice, we demonstrated that PTPN22 deficiency significantly shortened tail-bleeding time and accelerated arterial thrombus formation without affecting venous thrombosis and the coagulation factor VIII and IX. Consistently, PTPN22-deficient platelets exhibited enhanced platelet aggregation, granules secretion, calcium mobilization, lamellipodia formation, spreading and clot retraction. Quantitative phosphoproteomic analysis revealed the significant difference of phosphodiesterase 5A (PDE5A) phosphorylation in PTPN22-deficient platelets compared to wild-type platelets after collagen-related peptide (CRP) stimulation, which was confirmed by increased PDE5A phosphorylation (Ser92) in CRP-treated PTPN22-deficient platelets, concomitant with reduced cGMP level and VASP phosphorylation (Ser157/239). In addition, PTPN22 interacted with phosphorylated PDE5A (Ser92) and dephosphorylated it in activated platelets. Moreover, purified PTPN22 but not mutant form (C227S) possesses intrinsic serine phosphatase activity. Furthermore, inhibition of PTPN22 enhanced human platelet aggregation, spreading, clot retraction and increased PDE5A phosphorylation (Ser92). In conclusion, our study demonstrates a novel role of PTPN22 in platelet function and arterial thrombosis, identifying new potential targets for future prevention of thrombotic or cardiovascular diseases.