Abstract Mast Cells (MCs) initiate allergic responses but also participate in innate inflammatory reactions triggered by infection, tissue damage and cancer. MCs are recruited to affected sites where they release preformed and neo-formed mediators of inflammation. Bioactive lipids (such as sphingosine-1-phosphate, S1P) have demonstrated to induce MC chemotaxis. Recently, α-lysophosphatidilinositol (LPI) has awakened scientific interest due to the discovery of its endogenous receptor, the orphan receptor GPCR55. This receptor has been shown to couple to Gα12/13 pathway, controlling actin polimeryzation and cell migration. In this study, we aimed to characterize the role of LPI in the activation and migration of MCs. Using murine bone marrow-derived MCs, we observed that low concentrations of this lipid (10 nM and 100 nM) induces maximal ERK 1/2 phosphorylation at 15 and 10 minutes, respectively. Also, we found that LIMK (an important G12/13 and Rho-dependent kinase) is phosphorylated after 5 and 10 min after 10 nM or 100 nM LPI, respectively. Both ERK 1/2 and LIMK phosphorylation patterns were reproduced with the selective agonist of GPCR55, O-1602, at equi-potent concentrations to those of the LPI. Using Boyden chamber assays, we observed that LPI provocked higher chemotaxis of MCs when compared with S1P. Finally, using confocal microscopy, we observed that LPI triggered depolymerization of cortical actin, as well as the formation of filopodia between 15 and 30 minutes post stimulation. We concluded that GPR55 expressed in MCs induces important changes on MAPK and LIMK activation and its ligand, LPI, is a potent chemotactic factor of MCs.