The endogenous phospholipid l‐α‐lyso‐phosphatidylinositol (LPI) was recently identified as a novel ligand for the orphan G protein‐coupled receptor 55 (GPR55). In this study we define the downstream signaling pathways activated by LPI in a human embryonic kidney (HEK) 293 cell line engineered to stably express recombinant human GPR55. We find that treatment with LPI induces marked GPR55 internalization and stimulates a sustained, oscillatory Ca²⁺ release pathway, which is dependent on Gα13 and requires RhoA activation. We then establish that this signaling cascade leads to the efficient activation of NFAT (nu‐clear factor of activated T cells) family transcription factors and their nuclear translocation. Analysis of cannabinoid ligand activity at GPR55 revealed no clear effect of the endocannabinoids anandamide and 2‐arachidonoylglycerol;however, the classical CB₁ antagonist AM251 evoked GPR55‐mediated Ca²⁺ signaling. Thus, LPI is a potent and efficacious ligand at GPR55, which is likely to be a key plasma membrane mediator of LPI‐mediated signaling events and changes in gene expression.—Henstridge, C. M., Balenga, N. A. B., Ford, L. A., Ross, R. A., Waldhoer, M., Irving, A. J. The GPR55 ligand l‐α‐lysophosphatidylinositol promotes RhoA‐dependent Ca²⁺ signaling and NFAT activation. FASEB J. 23, 183‐193 (2009)