Objective To study the effects of melatonin (MT) on mitochondrial autophagy in neonatal rats with hypoxic-ischemic brain damage (HIBD).Method Animal model of HIBD was established.Forty-five 7-day-old Sprague-Dawley (SD) rats were randomly assigned to sham operation group and HIBD group.Brain tissue were taken at 0,2,4,6,8,12,24 and 48 h after model preparation,and the expressions of mitochondrial autophagy-related protein Bnip3 and autophagy-related protein LC3-Ⅱ were detected.Seventy-two 7-day-old SD rats were randomly assigned to sham operation group,HIBD group and post-HIBD treatment group (3-MA,Mdivi-I,Rapa,MT,3-MA + MT,Mdivi-1 + MT,Rapa + MT).The sizes of cerebral infarction after different treatment were detected using triphenyltetrazolium chloride staining (TIC).Primary cortical cells of fetal SD rats (embryonic day:17 ～ 19 d) were cultured.JC-I staining was used to detect mitochondrial membrane potential and immunofluorescence method was used to observe mitochondrial autophagy.The Oxygen glucose deprivation/reperfusion/R (OGD) model was prepared.Autophagy inhibitor 3-MA,mitochondrial autophagy inhibitor Mdivi-1,autophagy activator Rapa,and MT were applied and Bnip3 and LC3-Ⅱ expressions and CCK8 (Cell Counting Kit CCK 8) for cell viability assay were examined.Result TTC staining results showed significant white infarcts in the tissue of HIBD group after hypoxia-ischemia,especially in the 3-MA and Mdivi-1 groups,and the infarcts were smaller in Rapa group and groups with MT treatment,the differences were statistical significant (P ＜ 0.05).Compared with the sham operation group,the expressions of Bnip3 and LC3-Ⅱ in the HIBD group were significantly increased (P ＜ 0.05).Compared with the normal group,the expressions of Bnip3 and LC3-Ⅱ in the OGD/R group were increased (P ＜0.05).The activities of 3-MA and Mdivi-1 cells decreased significantly,the mitochondrial membrane potential decreased,and mitochondrial autophagy were decreased (P ＜ 0.05).The cell activity,mitochondrial membrane potential,and mitochondrial autophagy of Rapa group were increased (P ＜ 0.05).The cell viability,Bnip3 and LC3-Ⅱ expressions were increased in groups with MT intervention (P ＜ 0.05).Conclusion MT may play an important protective role in the early stage of brain injury by enhancing mitochondrial autophagy of HIBD,which provide a theoretical basis for the study of specific related mechanisms.