Objective: To investigate the effects of OX40 ligand(OX40L) reverse signaling on the expression and activity of matrix metalloproteinase-9 (MMP-9) in the cultured mouse aortic endothelial cells (MAECs). Methods: Aortic endothelial cells were isolated from C57BL/6J mice aged 8 to 10 weeks and cultured to the second passage for experiments. The cultured MAECs were divided into 4 groups:control group,sOX40 group,sOX40+anti-OX40L group and sOX40+Bapta-AM group,and stimulated with DMEM,soluble OX40,anti-OX40L monoclonal antibody and calcium chelator Bapta-AM,respectively. The mRNA and protein expressions of MMP-9 were determined by RT-PCR and Western blot. The activity of MMP-9 was assayed by zymography. The intracellular calcium ([Ca2+]i) in MAECs was detected by calcium imaging using fura-2. Results: A significant increase in intracellular calcium level of MAECs was observed after stimulation with soluble OX40 (0.1 mg/L) for 48 hours compared to the control,and MMP-9 mRNA and protein levels were also markedly elevated. The activity changes of MMP-9 in cell culture supernatant was consistent with that of intracellular level. Notably,these elevations can be blocked by soluble anti-OX40L monoclonal antibody(0.1 mg/L) or Bapta-AM(20 μmod/L). Conclusion:The stimulation of OX40L reverse signaling can activate the expression of MMP-9 on both mRNA and protein levels through the elevation of intracellular calcium of MAECs,suggesting that the OX40/OX40L signaling pathway may be one of the pathways contributing to the MMP-9 elevation in progression of atherosclerosis.