To explore in vitro the effect and mechanism of dimethyl fumarate (DMF) on modulating microglial polarization under chronic hypoperfusion-induced white matter injury (WMI). Methods: An in vitro model of low perfusion white matter demyelination was established by glucose-oxygen deprivation and stimulation with exogenous myelin debris, combined with DMF intervention in microglial cells. Real-time quantitative PCR, transcriptome sequencing, and immunofluorescence staining were employed to investigate the inflammatory phenotype transformation of microglia. Results: (1) Compared to the control group, the expression of pro-inflammatory genes interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α significantly decreased after DMF administration (P<0.05); (2) Transcriptome analysis revealed downregulation of neuroinflammation-related pathways and upregulation of autophagy-related pathways in microglia after DMF treatment; (3) Compared to the control group, the expression of autophagy-related genes and proteins was significantly increased in the DMF intervention group (P<0.05). Conclusion: DMF may regulate the transformation of microglia from a pro-inflammatory to an anti-inflammatory phenotype by activating the autophagy pathway.