BACKGROUND: Breastmilk stem cells (BSCs) have been reported to have potential benefits for infants. However, whether the BSCs could improve brain injury is unknown. A culture system for BSCs was established, and the roles of BSCs in treating white matter injury (WMI) were investigated in our study. METHODS: Breastmilk samples were collected from healthy lactating women between days 1 and 5 after delivery. The BSCs were cultured in a specialized culture medium and then characterized through flow cytometry and immunofluorescence methods. A rat model with WMI was established by ligating the right carotid artery of Sprague-Dawley rats at postnatal day 3 (P3) and exposing the rats to 6% hypoxia for 2 h. Rats were categorized into sham, WMI with breastmilk cell (WMI + BC), and WMI with (WMI + NS) groups. In the WMI + BC group, 5 µL BCs (1 × 10 RESULTS: The isolated BSCs expressed mesenchymal stem cell-positive markers, including CD105, CD73, CD29, CD166, CD44, and CD90. Meanwhile, the mesenchymal stem cell-negative markers, including HLA-DR, CD45, and CD79a, were also found in BSCs. The BSCs did not express pluripotent stem cell markers, including SOX2, Nanog, OCT4, SSEA4, and TRA-1-60. Immunofluorescence detection showed that BSCs expressed neural stem/progenitor cell markers, including Vimentin, Nestin, and A2B5. Following BSC treatment, pathological improvements were observed in WMI. The expressions of mature OLs markers myelin basic protein and myelin-associated glycoprotein were increased in the corpus callosum and periventricular areas. Meanwhile, the numbers of myelin sheath increased, and learning and memory abilities improved. Furthermore, a decrease in B7-2+/Iba1 + proinflammatory microglia and an increase in CD206+/Iba1 + anti-inflammatory microglia were observed. The mRNA expressions of proinflammatory factors (Il1b, Il6, Ifng, and Tnfa) and anti-inflammatory factors (Arg1 and Tgfb) decreased and increased, respectively. CONCLUSION: Our findings suggest that BSCs can improve the maturation of OLs following WMI in newborn rats. The mechanisms may be attributed to the reduced proinflammatory microglia cells and factors as well as the increased anti-inflammatory microglia cells and factors.