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Rhodiola crenulata is clinically used to combat hypobaric hypoxia brain injury at high altitude with the function of invigorating Qi and promoting blood circulation in Tibetan medicine. Salidroside (Sal), an active compound identified from Rhodiola species, has been shown to exert neuroprotective effects against hypoxic brain injury. However, its mitochondrial protective mechanisms remain largely unknown. The present study aimed to explore the mitochondrial protection of Sal and the involved mechanisms related to mitochondrial dynamics homeostasis on hypoxia-induced injury of HT22 cells. Hypoxic condition was performed as cells cultured in a tri-gas incubator with 1% O 2 , 5% CO 2 and 94% N 2. We firstly investigated the effects of different concentrations of Sal on the viability of normal or hypoxic HT22 cells. Whereafter, the levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), adenosine triphosphate (ATP) and Na+-K+-ATPase were tested by commercial kits. Meanwhile, mitochondrial superoxide, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were determined by specific labeled probes. Mitochondrial morphology was detected by mito-tracker green with confocal microscopy. Additionally, the potential interactions of Sal with Sirt1/p53/Drp1 signaling pathway-related proteins were predicted and tested by molecular docking and localized surface plasmon resonance (LSPR) techniques, respectively. Furthermore, the protein levels of Sirt1, p53, ac-p53, Drp1, p-Drp1(s616), Fis1 and Mfn2 were estimated by western blot analysis. Sal alleviated hypoxia-induced oxidative stress in HT22 cells as evidenced by increased cell viability and SOD activity, while decreased LDH release and MDA content. The protected mitochondrial function by Sal treatment was indicated by the increases of ATP level, Na+-K+-ATPase activity and MMP. Miraculously, Sal reduced hypoxia-induced mitochondrial fission, while increased mitochondrial tubular or linear morphology. The results of molecular docking and LSPR confirmed the potential binding of Sal to proteins Sirt1, p53, Fis1 and Mfn2 with affinity values 1.38 × 10−2, 5.26 × 10−3, 6.46 × 10−3 and 7.26 × 10−3 KD, respectively. And western blot analysis further demonstrated that Sal memorably raised the levels of Sirt1 and Mfn2, while decreased the levels of ac-p53, Drp1, p-Drp1 (s616) and Fis1. Collectively, our data confirm that Sal can maintain mitochondrial dynamics homeostasis by activating the Sirt1/p53/Drp1 signaling pathway. [Display omitted] [ABSTRACT FROM AUTHOR]