Interstitial cystitis/bladder pain syndrome (IC) is a debilitating condition of chronic pelvic pain with unknown etiology. Recently, we used a genetic approach in a murine model of IC to identify the lipase acyloxyacyl hydrolase (AOAH) as a modulator of pelvic pain. We found that AOAH-deficient mice have elevated pelvic pain responses, and AOAH immunoreactivity was detected along the bladder-brain axis. Lipidomic analyses identified arachidonic acid (AA) and its metabolite PGE2 as significantly elevated in the sacral spinal cord of AOAH-deficient mice, suggesting AA is a substrate for AOAH. Here, we quantified the effects of AOAH on phospholipids containing AA. Spinal cord lipidomics revealed increased AA-containing phosphatidylcholine in AOAH-deficient mice and concomitantly decreased AA-phosphatidylethanolamine, consistent with decreased CoA-independent transferase activity (CoIT). Overexpression of AOAH in cell cultures similarly altered distribution of AA in phospholipid pools, promoted AA incorporation, and resulted in decreased membrane fluidity. Finally, administration of a PGE2 receptor antagonist reduced pelvic pain in AOAH-deficient mice. Together, these findings suggest that AOAH represents a potential CoA-independent AA transferase that modulates CNS pain pathways at the level of phospholipid metabolism.