Prostaglandin E 2 (PGE 2) is an important mediator of the inflammatory response. Phospho-ceramide analogue-1 (PCERA-1), a synthetic phospholipid-like molecule, was previously reported to modulate pro- and anti-inflammatory cytokine production. We show here that PCERA-1 inhibited LPS-stimulated PGE 2 production in RAW264.7 macrophages, without affecting COX-2 expression. Furthermore, PCERA-1 efficiently suppressed arachidonic acid (AA) release in response to LPS. The dephosphorylated derivative of PCERA-1, ceramide analogue-1 (CERA-1), mimicked the inhibitory effect of PCERA-1 on AA release and PGE 2 production in macrophages. Inhibition of PGE 2 production by CERA-1 was completely rescued by addition of exogenous AA. Importantly, PCERA-1 and ceramide-1-phosphate (C1P) stimulated the enzymatic activity of cPLA 2α in an in vitro assay, whereas CERA-1 and ceramide inhibited both basal and C1P-stimulated cPLA 2α activity. Collectively, these results indicate that CERA-1 suppresses AA release and subsequent PGE 2 production in LPS-stimulated macrophages by direct interaction with cPLA 2, and suggest that ceramide may similarly counteract C1P effect on cPLA 2 activity in cells. The suppression of PGE 2 production is suggested to contribute to the anti-inflammatory action of PCERA-1.