Platelet hyperactivity is essential for thrombus formation in coronary artery diseases (CAD). Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients with cystic fibrosis elevates intracellular Cl- levels ([Cl-](i)) and enhanced platelet hyperactivity. In this study, we explored whether alteration of [Cl-](i) has a pathological role in regulating platelet hyperactivity and arterial thrombosis formation. CFTR expression was significantly decreased, while [Cl-](i) was increased in platelets from CAD patients. In a FeCl3-induced mouse mesenteric arteriole thrombosis model, platelet-specific Cftr-knockout and/or pre-administration of ion channel inhibitor CFTRinh-172 increased platelet [Cl-](i), which accelerated thrombus formation, enhanced platelet aggregation and ATP release, and increased P2Y(12) and PAR4 expression in platelets. Conversely, Cftr-overexpressing platelets resulted in subnormal [Cl-](i), thereby decreasing thrombosis formation. Our results showed that clamping [Cl-](i) at high levels or Cftr deficiency-induced [Cl-](i) increasement dramatically augmented phosphorylation (Ser422) of serum and glucocorticoid-regulated kinase (SGK1), subsequently upregulated P2Y(12) and PAR4 expression via NF-kappa B signaling. Constitutively active mutant S422(D) SGK1 markedly increased P2Y(12) and PAR4 expression. The specific SGK1 inhibitor GSK-650394 decreased platelet aggregation in wildtype and platelet-specific Cftr knockout mice, and platelet SGK1 phosphorylation was observed in line with increased [Cl-](i) and decreased CFTR expression in CAD patients. Co-transfection of S422(D) SGK1 and adenovirus-induced CFTR overexpression in MEG-01 cells restored platelet activation signaling cascade. Our results suggest that [Cl-](i) is a novel positive regulator of platelet activation and arterial thrombus formation via the activation of a [Cl-](i)-sensitive SGK1 signaling pathway. Therefore, [Cl-](i) in platelets is a novel potential biomarker for platelet hyperactivity, and CFTR may be a potential therapeutic target for platelet activation in CAD.