AbstractCrotoxin B (CB) is a catalytically active group IIA sPLA₂ from Crotalus durissus terrificus snake venom. In contrast to most GIIA sPLA₂s, CB exhibits anti-inflammatory effects, including the ability to inhibit leukocyte functions. Lipid droplets (LDs) are lipid-rich organelles associated with inflammation and recognized as a site for the synthesis of inflammatory lipid mediators. Here, the ability of CB to induce formation of LDs and the mechanisms involved in this effect were investigated in isolated macrophages. The profile of CB-induced 15-d-PGJ₂ (15-Deoxy-Delta-12,14-prostaglandin J₂) production and involvement of LDs in 15-d-PGJ₂ biosynthesis were also investigated. Stimulation of murine macrophages with CB induced increased number of LDs and release of 15-d-PGJ₂. LDs induced by CB were associated to PLIN2 recruitment and expression and required activation of PKC, PI3K, MEK1/2, JNK, iPLA₂ and PLD. Both 15-d-PGJ₂ and COX-1 were found in CB-induced LDs indicating that LDs contribute to the inhibitory effects of CB by acting as platform for synthesis of 15-d-PGJ₂, a pro-resolving lipid mediator. Together, our data indicate that an immunomodulatory GIIA sPLA₂ can directly induce LD formation and production of a pro-resolving mediator in an inflammatory cell and afford new insights into the roles of LDs in resolution of inflammatory processes.