Mutations in the GBA1 gene are common genetic risk factors for Parkinson's disease, disrupting enzymatic activity and causing lysosomal dysfunction, leading to elevated α-synuclein levels. Although the role of GBA1 in synucleinopathy is well established, recent research underscores neuroinflammation as a significant pathogenic mechanism in GBA1 deficiency. This study investigates neuroinflammation in Gba1 E326K knock-in mice, a model associated with increased risk of Parkinson's disease and dementia. At 9 and 24 months, we assessed GBA1 protein and activity, α-synuclein pathology, neurodegeneration, motor deficits and gliosis in the ventral midbrain and hippocampus using immunohistochemistry, western blot and glucocerebrosidase assays. Additionally, primary microglia from wild-type and Gba1E326K/E326K mice were treated with α-synuclein preformed fibrils to study microglia activation, pro-inflammatory cytokines, reactive astrocyte formation and neuronal death through quantitative PCR, western blot and immunocytochemistry analyses. We also evaluated the effects of gut inoculation of α-synuclein preformed fibrils in Gba1 E326K mice at 7 months and striatal inoculation at 10 months after injection, assessing motor/non-motor symptoms, α-synuclein pathology, neuroinflammation, gliosis and neurodegeneration via behavioural tests, immunohistochemistry and western blot assays. At 24 months, Gba1 E326K knock-in mice showed reduced glucocerebrosidase enzymatic activity and glucosylceramide build-up in the ventral midbrain and hippocampus. Increased pro-inflammatory cytokines and reactive astrocytes were observed in microglia and astrocytes from Gba1 E326K mice treated with pathological α-synuclein preformed fibrils. Gut inoculation of α-synuclein preformed fibrils increased Lewy body accumulation in the hippocampal dentate gyrus, with heightened microglia and astrocyte activation and worsened non-motor symptoms. Intrastriatal injection of α-synuclein preformed fibrils induced motor deficits, reactive glial protein accumulation and tauopathy in the prefrontal cortex and hippocampus of Gba1 E326K mice. GBA1 deficiency attributable to the Gba1 E326K mutation exacerbates neuroinflammation and promotes pathogenic α-synuclein transmission, intensifying disease pathology in Parkinson's disease models. This study enhances our understanding of how the Gba1 E326K mutation contributes to neuroinflammation and the spread of pathogenic α-synuclein in the brain, suggesting new therapeutic strategies for Parkinson's disease and related synucleinopathies.