Increased glomerular prostaglandin E₂ (PGE₂) production is associated with the progression of diseases such as membranous nephropathy, nephrotic syndrome, and anti-Thy1 nephritis. We investigated the signaling pathways that regulate the synthesis and actions of PGE₂ in glomerular podocytes. To study its actions, we assessed the ability of PGE₂ to regulate the production of its own precursor, arachidonic acid (AA), in a mouse podocyte cell line. PGE₂ dose-dependently reduced phorbol ester (PMA)-mediated AA release. Inhibition of PMA-stimulated AA release by PGE₂ was found to be cAMP/PKA-dependent, because PGE₂ significantly increased levels of this second messenger, whereas the inhibitory actions of PGE₂ were reversed by PKA inhibition and reproduced by the cAMP-elevating agents forskolin and IBMX. PGE₂ synthesis in this podocyte cell line increased fourfold at 60 min in response to PMA, coinciding with upregulation of cyclooxygenase (COX)-2 but not COX-1 levels. However, PGE₂ synthesis was significantly reduced by COX-1-selective inhibition, yet to a lesser extent by COX-2-selective inhibition. Our findings suggest that PMA-stimulated PGE₂ synthesis in mouse podocytes requires both basal COX-1 activity and induced COX-2 expression, and that PGE₂ reduces PMA-stimulated AA release in a cAMP/PKA-dependent manner. Such an autocrine regulatory loop might have important consequences for podocyte and glomerular function in the context of renal diseases involving PGE₂ synthesis.