Objective To observe the effect of human umbilical cord mesenchymal stem cell-derived exosomes （HUC-MSC-Exo） on microglial polarization in neonatal rats with white matter injury （WMI）. Methods Three-day-old Sprague-Dawley rats were randomly divided into sham group （Sham）， hypoxia-ischemia group （HI） and HUC-MSC-Exo group， with 12 rats in each group. Unilateral common carotid artery ligation combined with hypoxia （8% oxygen and 92% nitrogen） was used to construct a WMI rat model. Exosomes were extracted by ultra-high-speed centrifugation and characterized by nanoflow cytometry， western blot experiments and transmission electron microscopy. Brain stereotaxic-assisted inferior ventricular transplantation exo （2×10⁸ particles/μL） was performed and brain tissue samples were collected 14 days after HI. Hematoxylin-eosin （HE） staining was used to observe morphological changes of brain tissue. Nissl staining was used to observe Nissl body formation in brain tissue； Luxol fast blue （LFB） staining was used to observe the formation of myelin sheath in brain tissue. Immunofluorescence staining was used to observe the localized expression of ionic calcium binds adaptor molecule 1 （Iba1）. The protein expression levels of cluster of differentiation 86 （CD86）， inducible nitric oxide synthase （iNOS）， tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， CD206， arginase-1 （Arg-1）， IL-10 and transforming growth factor-β （TGF-β） were detected by western blot. Results The results of nanoflow cytometry， western blot and transmission electron microscopy showed that the diameter of HUC-MSC-Exo particles was between 30 and 150 nm， and the oval-like shape and membrane-like structure were visible， and the exo markers CD9， CD63 and TSG101 were positive， while calnexin was negative. HE staining， Nissl staining and LFB staining showed that compared with the Sham group， the HI group had brain tissue structure destruction， which was manifested by cell morphological changes， nerve fiber arrangement disorder and vacuolation， Nissl body dissolution or even disappearance， and myelination was blocked. HUC-MSC-Exo significantly reversed the pathological changes in the HI group. The results of immunofluorescence staining and western blot showed that the microglial marker Iba1 was mainly expressed in the subventricular zone （SVZ）， and the expression of Iba1 protein in the SVZ region increased after HI compared with the Sham group （t = 15.95、20.31， P < 0.01）. HUC-MSC-Exo significantly reduced the expression of Iba1 protein in the HI group （t = 10.35、11.01， P < 0.01）. The results of western blot showed that the expressions of M1 microglia markers （CD86 and iNOS） and pro-inflammatory cytokines （TNF-α and IL-1β） were significantly increased after HI （t = 10.98、7.68、15.13、13.13， both P < 0.01）， and the expressions of M2 microglia markers （CD206 and Arg-1） and anti-inflammatory cytokines （IL-10 and TGF-β） were also increased after HI （t = 14.26、9.38、8.82、7.42， both P < 0.01）. HUC-MSC-Exo decreased the protein expression of CD86， iNOS， TNF-α and IL-1β （t = 9.79、5.81、8.06、7.03， all P < 0.01） and increased the protein expression levels of CD206， Arg-1， IL-10 and TGF-β compared to the HI group （t = 12.90、8.16、8.98、9.49， both P < 0.01）. Conclusion HUC-MSC-Exo attenuates WMI in neonatal rats by regulating microglial polarization.