Lysophosphatidylinositol (LPI) is an endogenous signaling molecule for the GPR55 receptor. Previous studies have shown that arachidonoyl-lysophosphatidylinositol (LPI-20:4) produced an increase in the inflammatory mediators NLPR3 (inflammasome-3 marker) and IL-1b in neurons from both rat dorsal root ganglion (DRG) and hippocampal cultures. Because LPI is comprised of a family of lipid structures that vary in fatty acyl composition, the current work examined neuroinflammatory responses to various LPI structures in DRG and hippocampal cultures as assessed by high-content fluorescent imaging. Major endogenous LPI fatty acyl structures consisting of 16:0, 18:0, 18:1, or 20:4 were compared for their effects on IL-1b, NLRP3, and GPR55 immunoreactive areas of neurites and cell bodies after a 6-h treatment. Among these four LPI structures, only LPI-20:4 treatment produced increases in immunoreactive areas for GPR55, NLRP3, and IL-1b in DRG and hippocampal neurites. In contrast, all other LPI structures tested produced a decrease in all of these inflammatory immunoreactive areas in both neurites and cell bodies. Additional studies with LPI-20:4 treatment indicated that IL-6, IL-18, and TNF-α were significantly increased in neurites of DRG and hippocampal cultures. However, oleoyl-lysophosphatidylinositol (LPI-18:1) treatment produced decreases in these three cytokines. Using the viability dye Alamar blue, LPI-20:4 was shown to produce concentration-dependent decreases, whereas all other endogenous LPI structures produced increases with this assay. These studies indicate that fatty acyl structure is the major determinant of LPI for neuroinflammatory responses in DRG and hippocampal cultures, with LPI-20:4 showing pro-inflammatory effects and all other endogenous LPIs tested exhibiting anti-inflammatory responses.