Human group IIA-secreted phospholipase A2 (sPLA2-IIA) is an important regulator of cytokine-mediated inflammatory responses in both in vitro and in vivo models of rheumatoid arthritis (RA). However, treatment of RA patients with sPLA2-IIA inhibitors shows only transient benefit. Using an activity-impaired sPLA2-IIA mutant protein (H48Q), we show that up-regulation of TNF-dependent PGE2 production and cyclooxygenase-2 (COX-2) induction by exogenous sPLA2-IIA in RA fibroblast-like synoviocytes (FLSs) is independent of its enzyme function. Selective cytosolic phospholipase A2-α (cPLA2-α) inhibitors abrogate TNF/sPLA2-IIA-mediated PGE2 production without affecting COX-2 levels, indicating arachidonic acid (AA) flux to COX-2 occurs exclusively through TNF-mediated activation of cPLA2-α. Nonetheless, exogenous sPLA2-IIA, but not H48Q, stimulates both AA mobilization from FLSs and microparticle-derived AA release that is not used for COX-2-dependent PGE2 production. sPLA2-IIA-mediated AA production is inhibited by pharmacological blockade of sPLA2-IIA but not cPLA2-α. Exogenous H48Q alone, like sPLA2-IIA, increases COX-2 protein levels without inducing PGE2 production. Unlike TNF, sPLA2-IIA alone does not rapidly mobilize NF-κB or activate phosphorylation of p38 MAPK, two key regulators of COX-2 protein expression, but does activate the ERK1/2 pathway. Thus, sPLA2-IIA regulates AA flux through the cPLA2-α/COX-2 pathway in RA FLSs by up-regulating steady state levels of these biosynthetic enzymes through an indirect mechanism, rather than direct provision of substrate to the pathway. Inhibitors that have been optimized for their potency in enzyme activity inhibition alone may not adequately block the activity-independent function of sPLA2-IIA.