Parkinson’s disease (PD) is a neurodegenerative disease characterized by the death of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies that are composed of aggregated α-synuclein (α-Syn). However, the factors that regulate α-Syn pathology and nigrostriatal dopaminergic degeneration remain poorly understood. Previous studies demonstrate cholesterol 24-hydroxylase (CYP46A1) increases the risk for PD. Moreover, 24-hydroxycholesterol (24-OHC), a brain-specific oxysterol that is catalyzed by CYP46A1, is elevated in the cerebrospinal fluid of PD patients. Herein, we show that the levels of CYP46A1 and 24-OHC are elevated in PD patients and increase with age in a mouse model. Overexpression of CYP46A1 intensifies α-Syn pathology, whereas genetic removal of CYP46A1 attenuates α-Syn neurotoxicity and nigrostriatal dopaminergic degeneration in the brain. Moreover, supplementation with exogenous 24-OHC exacerbates the mitochondrial dysfunction induced by α-Syn fibrils. Intracerebral injection of 24-OHC enhances the spread of α-Syn pathology and dopaminergic neurodegeneration via elevated X-box binding protein 1 (XBP1) and lymphocyte-activation gene 3 (LAG3) levels. Thus, elevated CYP46A1 and 24-OHC promote neurotoxicity and the spread of α-Syn via the XBP1–LAG3 axis. Strategies aimed at inhibiting the CYP46A1-24-OHC axis and LAG3 could hold promise as disease-modifying therapies for PD.