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Oxidative stress is a major risk factor that causes osteocyte cell death and bone loss. Prior studies primarily focus on the function of cell surface expressed Cx43 channels. Here, we reported a new role of mitochondrial Cx43 (mtCx43) and hemichannels (HCs) in modulating mitochondria homeostasis and function in bone osteocytes under oxidative stress. In osteocyte MLO-Y4 cells, the translocation of Cx43 to mitochondria was increased under H2O2-induced oxidative stress. H2O2 increased the mtCx43 level accompanied by elevated mitochondrial Cx43 HC activity determined by dye uptake assay. Cx43 knockdown (KD) by CRISPR-Cas9 lentivirus system resulted in impairment of mitochondrial function, primarily manifested as decreased ATP production. Cx43 KD had reduced intracellular reactive oxidative species (ROS) levels and mitochondrial membrane potential. Additionally, live-cell imaging results demonstrated that the proton flux was dependent upon mtCx43 HCs because its activity was specifically inhibited by an antibody targeting Cx43 C-terminus. The co-localization and interaction of mtCx43 and ATP synthase subunit F (ATP5J2) were confirmed by fluorescence resonance energy transfer (FRET) and a protein pull-down assay. Together, our study suggests that mtCx43 hemichannels regulate mitochondrial ATP generation by mediating K+, H+, and ATP transfer across the mitochondrial inner membrane and the interaction with mitochondrial ATP synthase, leading to enhancing the protection capacity of osteocytes against oxidative insults.