The regulated expression of ICAM-1 plays an important role in inflammatory processes and immune responses. The present study aimed to determine the in vivo involvement of cytosolic phospholipase A2α (cPLA2α) in ICAM-1 overexpression during inflammation and to elucidate the cPLA2α-specific role in signal events leading to ICAM-1 upregulation in endothelial cells. cPLA2α and ICAM-1 upregulation were detected in inflamed paws of mice with collagen-induced arthritis and in periepididymal adipose tissue of mice fed a high-fat diet. Intravenous injection of 2 mg/kg oligonucleotide antisense against cPLA2α (AS) that reduced cPLA2α upregulation also decreased ICAM-1 overexpression, suggesting a key role of cPLA2α in ICAM-1 upregulation during inflammation. Preincubation of endothelial ECV-304 cells that express ICAM-1 and of HUVEC that express ICAM-1 and VCAM-1 with 1 μM AS prevented cPLA2α and the adhesion molecule upregulation induced by TNF-α and inhibited their adherence to phagocyte like-PLB cells. Whereas AS did not inhibit NADPH oxidase 4-NADPH oxidase activity, inhibition of oxidase activity attenuated cPLA2α activation, suggesting that NADPH oxidase acts upstream to cPLA2α. Attenuating cPLA2α activation by AS or diphenylene iodonium prevented the induction of cyclooxygenase-2 and the production of PGE2 that were essential for ICAM-1 upregulation. Inhibition of cPLA2α activity by AS inhibited the phosphorylation of both p65 NF-κB on Ser536 and protein kinase A-dependent CREB. To our knowledge, our results are the first to show that CREB activation is involved in ICAM-1 upregulation and suggest that cPLA2α activated by NADPH oxidase is required for sequential phosphorylation of NF-κB by an undefined kinase and CREB activation by PGE2-mediated protein kinase A.