Eicosanoids are potent mediators of inflammatory response whose role has been well established in inflammatory disorders. Release of arachidonic acid by group IVA cytosolic phospholipase A2 α (cPLA2α) is the initial rate limiting step for the production of eicosonoids in response to inflammatory mediators. Previous findings from our laboratory have demonstrated that cPLA2α is directly activated by the emerging bioactive sphingolipid, ceramide-1-phosphate (C1P). In this study, we have developed a modified Triton X-100/phosphatidylcholine (PC) mixed micelle assay which was utilized to determine the kinetics and specificity of this lipid-enzyme interaction. Using this assay, the activity of the enzyme increased in a dose dependent manner with increasing amount of C1P in the mixed micelle and the stoichiometry of this interaction was found to be 2 molecules of C1P to achieve full activation. This activation was found to be lipid specific as other phospholipids such as PE, PS, PA, DAG, and S1P had insignificant effect on cPLA2α activity. Furthermore, based on previous studies we hypothesized that the specific interaction site for C1P was localized to the cationic β-groove (R57, K58, R59) of the C2 domain of cPLA2α. In this regard, mutants of this region of cPLA2α were generated ((R57A/K58A/R59A), (R57A/R59A), (K58A/R59A), (R57A/K58A), (R57A), (K58A), and (R59A)) and examined for C1P affinity by surface plasmon resonance (SPR). The triple, the double mutants, and the single mutant (R59A) demonstrated significantly reduced affinity for C1P containing vesicles compared to wild-type cPLA2α. Examining these five mutants for enzymatic activity demonstrated significant reduction in the ability of C1P to increase the Vmax of the reaction and significantly decreased the dissociation constant (KSA) of the reaction as compared to the wild-type enzyme. The mutational effect was specific for C1P as all of the cationic mutants of cPLA2α demonstrated normal basal activity as well as normal affinities for PC and PtdIns(4,5)P2 compared to wild-type cPLA2α. Finally, we demonstrated these amino acids were critical for translocation of cPLA2α in A549 lung adenocarcinoma cells in response to inflammatory agonists like A23187 and IL-1β. Lastly, we also demonstrated the mechanistic difference between activation of cPLA2α by the two anionic lipids, C1P and PI(4,5)P2.