Objective To investigate mitochondrial function mediated by phospholipase D1 (PLD1) in the lungs of mice with bronchopulmonary dysplasia. Methods Wild-type ( WT) and PLD1 knockout ( PLD1-KO)newborn mice were assigned to four groups: normoxic+WT, normoxic+PLD1-KO, hyperoxic+WT, and hyperoxic+ PLD1-KO, with nine mice in each group. Mice in the hyperoxia groups were exposed to hyperoxia (85% O₂ ) for 14 days. Mice in the normoxic groups were exposed to normoxic conditions (21% O₂ ) for 14 days. On the 14th day, the levels of oxidative stress, apoptosis, and fibrosis in lungs were evaluated using commercial kits for malondialdehyde (MDA) and superoxide dismutase ( SOD ), Western blot for BAX, BCL-2, and Cleaved Caspase-3, and immunohistochemistry for α-SMA and AIF. The following MLE-12 cell groups were prepared, normoxic + si-NC,hyperoxic+si-NC, normoxic+si-PLD1, and hyperoxic+si-PLD1. After transient transfection, the cells were exposed to normoxia or hyperoxia for 24 h. Mitochondrial reactive oxygen species (mtROS) and function were measured using MitoSOX Red and the hippocampus mitochondrial stress test. Results The levels of α-SMA and AIF staining, MDA,Cleaved Caspase 3, and BAX in lung tissue were significantly increased in the hyperoxic groups compared with the normoxic groups (P<0. 05), while SOD activity and BCL-2 levels were significantly decreased (P<0. 05). α-SMA and AIF staining, and the abundance of Cleaved Caspase-3 and BAX in lung tissue were lower in the hyperoxia+ PLD1-KO group than in the hyperoxia+WT group (P<0. 05), while SOD activity and BCL-2 abundance were higher in the hyperoxia+PLD1-KO group than in the hyperoxia+WT group (P< 0. 05). The level of AIF in MLE-12 cell mitochondria in the hyperoxic groups was significantly lower than that in the normoxic groups (P<0. 05); however, the level of AIF was increased significantly in the cytoplasm of the hyperoxic groups compared with the normoxic groups (P<0. 05). The level of AIF in MLE-12 cell mitochondria in the hyperoxic+si-PLD1 group was significantly increased compared with that in the hyperoxic+si-NC group (P<0. 05). The abundance of mtROS in hyperoxia MLE- 12 cell groups was higher than that in the normoxia groups (P<0. 05), and the abundance of mtROS in the hyperoxia +si-PLD1 group was lower than that in the hyperoxia+si-NC group (P<0. 05). Compared with the normoxic+si-NC group, basic respiration, ATP production, maximum respiration, and spare respiratory capacity was significantly decreased in the hyperoxic+si-NC group (P<0. 05). Compared with the hyperoxic+si-NC group, the hyperoxic+siPLD1 group had significantly increased basic respiration, ATP production, maximum respiration, and spare respiratory capacity ( P<0. 05). Conclusions PLD1 is involved in hyperoxia-induced injury of mouse BPD and MLE-12 cells. Deletion of the PLD1 gene may alleviate hyperoxia-induced lung injury by inhibiting mitochondrialdependent apoptosis.