Our earlier in vitro and in vivo studies have revealed that the phytosterol, pentalinonsterol (cholest-4,20,24-trien-3-one) (PEN), isolated from the roots of Pentalinon andrieuxii, possesss immunomodulatory properties in macrophages and dendritic cells. Leishmaniasis, caused by the infection of Leishmania spp. (a protozoan parasite), is emerging as the second-leading cause of mortality among the tropical diseases and there is an unmet need for a pharmacological intervention of leishmaniasis. Given the beneficial immunomodulatory actions and lipophilic properties of PEN, the objective of this study was to elucidate the mechanism(s) of action of the immunomodulatory action(s) of PEN in macrophages through the modulation of phospholipase A (PLA) activity that might be crucial in the antileishmanial action of PEN. Therefore, in this study, we investigated whether PEN would modulate the activity of PLA in RAW 264.7 macrophages and mouse bone marrow-derived primary macrophages (BMDMs) in vitro and further determined how the upstream PLA activation would regulate the downstream cytokine release in the macrophages. Our current results demonstrated that (i) PEN induced PLA activation (arachidonic acid release) in a dose- and time-dependent manner that was regulated upstream by the mitogen-activated protein kinases (MAPKs); (ii) the PEN-induced activation of PLA was attenuated by the cPLA-specific pharmacological inhibitors; and (iii) the cPLA-specific pharmacological inhibitors attenuated the release of inflammatory cytokines from the macrophages. For the first time, our current study demonstrated that PEN exhibited its immunomodulatory actions through the activation of cPLA in the macrophages, which potentially could be used in the development of a pharmacological intervention against leishmaniasis.