BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) brain damage is related to inflammatory responses and oxidative stress. Interleukin (IL)-35 is an antioxidant and anti-inflammatory cytokine. Thus, the effect of IL-35 treatment on neonatal rats with hypoxic-ischemic brain injury was investigated. METHODS: A total of 96 7-day-old Sprague Dawley rats were randomly divided into three groups: sham group, HIE group, and IL-35 group. After left common carotid occlusion and 2.5 h hypoxia (HI injury), IL-35 (20 µg/g) was intraperitoneally (i.p.) administered to the pups. In vitro, BV2 cells were treated with or without IL-35 6 h before oxygen-glucose deprivation (OGD) insult and the microglia culture medium (MCM) was co-cultured with b.End3 cerebral vascular endothelial cells. Microglial polarization and activation were assessed by real-time quantitative polymerase chain reaction (RT-qPCR), Western blot, and enzyme-linked immunosorbent assay (ELISA). Endothelial cell dysfunction was measured by cell counting kit-8 and Western blot assays. RESULTS: Administration of IL-35 alleviated neurological deficiencies, decreased brain edema, ameliorated cerebral infarction, and limited M1 microglial polarization in HI-injured pups. Meanwhile, IL-35 decreased pro-inflammatory cytokines, tumor necrosis factor-α, IL-1β, and reactive oxygen species generation in OGD-induced bEnd.3 cells. Furthermore, IL-35 treatment could reverse the vascular endothelial cell injury induced by microglial polarization. Finally, IL-35 markedly suppressed the activation of hypoxia-inducible factor-1α (HIF-1α) and the nuclear factor-κB (NF-κB) signaling pathway in vivo and in vitro. CONCLUSIONS: IL-35 relieved hypoxic-ischemic-induced brain injury and inhibited the inflammatory response by suppressing microglial polarization and activation. These results suggest that IL-35 might have potential applications for the treatment of HIE.