The ways in which diet and sex influence isoform-specific phospholipase A 2 (PLA 2) mediated fatty acid release and oxylipin formation are underexplored, particularly with respect to oxylipin formation. This is relevant to the use of PLA 2 inhibitors as potential anti-inflammatory drugs in cardiovascular disease. To determine diet and sex effects on PLA 2 activity, Sprague-Dawley rats received diets with 1.3% alpha-linolenic acid (ALA) or docosahexaenoic acid (DHA) for 6 weeks. Heart homogenates were incubated with varespladib (sPLA 2 inhibitor) or methyl arachidonyl fluorophosphonate (cPLA 2 /iPLA 2 inhibitor) and non-esterified PUFA and oxylipins were determined by HPLC-MS/MS. Data analysis using 3-way repeated measures ANOVA revealed that sPLA 2 activity was higher in females, and although sPLA 2 contributed to ~50% of arachidonic acid (ARA) release (a rationale for its anti-inflammatory role), it did not impact ARA oxylipin formation to the same extent, and it was selective for DHA release and DHA oxylipin formation. sPLA 2 also mediated eicosapentaenoic acid (EPA) release only in ALA fed rats. In contrast, cPLA 2 and/or iPLA 2 activity contributed majorly to EPA and ALA release, and the formation of their and linoleic acid oxylipins, although there was activity towards nearly all PUFA and oxylipin formation. cPLA 2 and/or iPLA 2 activity was also more often influenced by diet, with most activity towards ALA in ALA fed rats and DHA in DHA fed rats. Interestingly, stimulated heart ARA oxylipin formation via cyclooxygenase was markedly higher in males, but only when consuming the ALA diet. In conclusion, higher sPLA 2 in females and its selectivity for DHA and formation of anti-inflammatory DHA oxylipins, as well as the greater formation of pro-inflammatory ARA oxylipins via cyclooxygenase in stimulated hearts of males consuming no DHA, may contribute to our understanding of the (apparent lack of) cardiovascular benefit of sPLA 2 inhibitors and to sex differences in cardiovascular health.