Phloroglucinol is the monomeric units of phlorotannins abundant in brown algae. Several biological effects of phloroglucinol have been reported, however, antithrombotic and profibrinolytic activities of phloroglucinol have not been studied yet. In this study, the anticoagulant properties of phloroglucinol were determined by assays of activated partial thromboplastin time (aPTT), prothrombin time (PT) and cell based thrombin and activated factor X (FXa) generation activities. And the effects of phloroglucinol on the expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were tested in tumor necrosis factor-α (TNF-α) activated human endothelial cells (HUVECs). I found that phloroglucinol prolonged aPTT and PT significantly and inhibited thrombin and FXa generation in HUVECs. Furthermore, phloroglucinol inhibited TNF-α induced PAI-1 production. I then used pathway inhibitors to investigate which step of the TNF-α induced signaling pathway was targeted by phloroglucinol. I observed that the c-Jun N-terminal kinase (JNK) inhibitor increased the inhibitory effects of phloroglucinol, whereas the nuclear factor factor-κB (NF-κB) and the extracellular signal-regulated kinase (ERK) inhibitor did not. Therefore these results suggest that phloroglucinol possess antithrombotic and profibrinolytic activities and that NF-κB and ERK pathways are possible targets of phloroglucinol in the regulation of TNF-α stimulated PAI-1 production in HUVECs.