Background: The potential signaling pathway of TSA suppressing TF expression induced by thrombin was unknown. Thus, the transcription of TF in HUVECs and the expressions of DCF, phospho-p38 MAPK, NADPH oxidase 4, PAR-1, and NF-κB were detected in our study. Methods: HUVECs were randomly divided into control group, thrombin-treated group (with 5 U/mL of thrombin), and 4 TSA-treated groups (with 5 U/mL of thrombin plus TSA with 4 different concentrations of 1 μg/mL, 10 μg/mL, 100 μg/mL, and 1 mg/mL, respectively). Results: After incubation with thrombin for 6 h at 37°C, the results showed increased TF mRNA, TF procoagulant activity, and antigen of TF in HUVECs of thrombin-treated group (p < 0.01); however, they were restored by TSA in a dose-dependent manner (p < 0.01). In addition, reactive oxygen species (ROS), phospho-p38 MAPK, NADPH oxidase 4, NF-κB, and PAR-1 expressed more intensively, and phosphorylated Akt decreased obviously in HUVECs after thrombin stimulation (p < 0.01); however, they were reversed to different extents by TSA in a dose-dependent manner (p < 0.01). Conclusions: Study suggests that TSA inhibits TF expression induced by thrombin in cultured HUVECs, and the potential signaling pathway of which is TSA interrupts the activation of PAR-1 and NADPH oxidase as well as derivative ROS generation, thereafter suppresses the activation of NF-κB, the upstream signal molecule of TF, via hampering phosphorylation of p38 MAPK and dephosphorylation of Akt, and finally inhibits thrombin-induced TF overexpression.