Deep vein thrombosis (DVT) is a common condition that often leads to pulmonary thromboembolism and death. Cyclooxygenase (COX)-1 and -2 catalyze the formation of prostaglandins and thromboxane (TX)A2 from arachidonic acid and play a critical role in the thrombotic events, however, the impact of COX-2 deletion (COX-2KO) in the DVT remains unclear. In this study, traditional (thromboelastomethry, mass weight and histology) and innovative (venous ultrasonography) approaches were used to assess the effect of COX-2 ablation on DVT in mice. In particular, ligation of the inferior vena cava to induce thrombus formation in COX-2KO and WT mice was carried out. Thromboelastometry analyses performed in whole blood of COX-2KO revealed alterations of clotting factors, and of platelet activity, with consequent greater clot firmness and clot elasticity. COX-2KO mice have increased plasma levels of functional fibrinogen, factor VIII and tissue factor (TF), and higher TXA2 serum than WTs. Finally, PAI-1 activity was the highest in COX-2KO, whereas tPA activity was similar in the two groups. The propensity to thrombosis of COX-2KO mice was also confirmed by ultrasonography. Genetic deletion of COX-2 was associated with bigger thrombi already at early time point. At 48 hours COX-2KO thrombi showed a greater content of connective tissue and fibrin, and increased number of infiltrated monocytes compared to WT. Remarkably, Annexin A2 (ANXA2), a fibrinolytic receptor, was significantly increased in cells present in COX-2KO thrombi. In addition, peritoneal macrophages from COX-2KO mice showed increased expression of ANXA2 with different distribution at cellular level compared to WTs. In particular, ANXA2 was expressed in the cytoplasm and in the nucleus of COX-2KO macrophages, whereas in control cells the protein was found mainly localized on cell membrane/periphery. In conclusion, the increased basal activation of haemostatic system observed in COX-2KO mice may partly explain the predisposition of these mice to thrombosis. In addition, the different localization of ANXA2 in monocytes from COX-2KO mice with respect to wild type mice suggests that this protein may display additional protective, as yet unknown, mechanisms influencing the stability of venous thrombi.