AIM: White matter injury (WMI), characterized by white matter degeneration and iron deposition, contributes to neurological dysfunction. Histone deacetylase 3 (HDAC3) is implicated in neurodegenerative processes, yet its role in WMI-associated ferroptosis remains unclear. METHODS: Clinical assessments in WMI patients revealed correlations between serum iron, α-synuclein, and antioxidant levels and MRI-confirmed white matter degeneration. In a cuprizone-induced demyelination mouse model, white matter integrity, oligodendrocyte dysfunction, iron accumulation, and lipid peroxidation were evaluated through behavioral testing, histological staining, and biochemical analyses. To identify potential molecular targets of HDAC3-mediated ferroptosis, CUT&Tag sequencing was performed. The involvement of this pathway was further validated in vitro using iron overload assays and in vivo through HDAC3 overexpression via AAV vectors. RESULTS: In the present study, HDAC3 expression was elevated following demyelination and was suppressed by RGFP966 treatment. Brain MRI findings from clinical patients and histological analyses in CPZ-treated mice revealed disrupted iron metabolism following white matter injury, likely driven by increased iron deposition and lipid peroxidation in the affected regions. HDAC3 inhibition alleviated oligodendrocyte lineage dysfunction, preserved myelin integrity, and mitigated cognitive and motor deficits induced by demyelination. CUT&Tag sequencing suggested that the therapeutic effects of RGFP966 are mediated through HDAC3-dependent regulation of ferroptosis. The reliability of these findings was further supported by in vivo validation of ferroptosis-related gene expression, indicating that the HDAC3-pyruvate dehydrogenase kinase 4 (PDK4) axis plays a critical role in the epigenetic regulation of ferroptosis-related pathways. CONCLUSION: HDAC3 drives ferroptosis in WMI via iron metabolism and lipid peroxidation, highlighting the HDAC3-PDK4 axis as a therapeutic target.