The aim of the study was to investigate the effect of LPS-induced PreC on ischemic brain injury and characterize the mainly involved signaling pathways of microglia. Furthermore, we hypothesized that PI3Kγ regulates different microglial immune functions, critical for conveying the LPS-induced PreC effects upon ischemic brain injury. Experiments were performed in wild-type (WT), PI3Kγ knockout (PI3Kγ-/-) and PI3Kγ kinase-dead (PI3KγKD/KD) mice, which were either exposed to middle cerebral artery occlusion or not (Control). MCAO mice received a PreC stimulus (LPS i.p. injection; MCAO+PreC) or a control injection of saline (MCAO). Furthermore, microglia cells were isolated from mice brains after MCAO or MCAO+PreC and mass spectrometry analysis highlighted proteomic characteristics. Additionally, brain tissue was analyzed for microglial inflammatory responses via immunohistochemical workup. Our data revealed that LPS PreC attenuates the extent of ischemic brain injury and that neuroprotection is in consequence of the altered regulation of inflammatory microglial functions, uttered in a differential microglial proteome. Genotypic differences emerged upon LPS-induced protection, with wild-type mice, being protected the most (49%) in reducing infarct volume, PI3KγKD/KD-mice showing a moderate protection (35%) and PI3Kγ-/- mice exhibiting the lowest protection effect (21%). Furthermore, our study revealed a tight regulation of phagocytosis, migration and PMN expression upon LPS PreC by partly lipid kinase independent activities of PI3Kγ in microglial cells. Therefore, these results confirm and expand recent data on regulatory effects of PI3Kγ on microglial immune functions and induction of innate immune memory and underline the involvement of PI3Kγ in the disease model of ischemic brain injury.