Tissue factor (TF) is a core protein with an essential function in the coagulation cascade that maintains the homeostasis of the blood vessels. TF not only participates in neointima formation, but also causes the development of atherosclerosis. This study investigated the mechanism regulating TF expression in macrophages using Pam3CSK4, a TLR2 ligand. Pam3CSK4 induced TF expression in two types of macrophages (Raw264.7 and BMDM), but not in TLR2 KO mice derived BMDM. Pam3CSK4 induced TF expression was inhibited by pretreatment with pan-JAK inhibitor or JAK2 inhibitor AG490. JAK2 knock-down by siRNA inhibited Pam3CSK4 induced TF expression. Pam3CSK4 stimulated STAT3 phosphorylation (S727), while STAT3 knock-down by siRNA reduced Pam3CSK4 induced TF expression. These results suggest that Pam3CSK4 induced TF expression is regulated by the JAK2–STAT3 signaling pathway. Pam3CSK4, unlike increased TF expression, significantly decreased RGS2 expression, while RGS2 overexpression decreased Pam3CSK4 induced TF expression. Inhibition of TF by RGS2 WT did not occur in mutants with flawed RGS domains. We also investigated the correlation between RGS2 and STAT3 phosphorylation. RGS2 knock-down elevated Pam3CSK4 induced STAT3 phosphorylation, but RGS2 overexpression had the opposite effect on STAT3 phosphorylation. These results suggest that, while Pam3CSK4 induced TF expression is regulated by JAK2–STAT3 signaling, RGS2 is a negative regulator targeted to STAT3. J. Cell. Biochem. 114: 1315–1321, 2013. © 2012 Wiley Periodicals, Inc.