In order to find safe and effective anti-hypoxic molecules, a series of 25 novel salidroside derivatives R1~R10、 X1~X10 and J1~J5 were designed and synthesized using p-hydroxyphenylethanol and 1,2,3,4,6-β-D-glucosaccharide pentaacetate as starting materials. Their structures were confirmed by ¹H NMR, ¹³C NMR and HR-MS(ESI), which have not been reported in the literature. The anti-hypoxia activities of the target compounds were evaluated by the hypoxia/reoxygenation model of H9C2 cardiomyocytes in vitro, then the cytotoxicities of R7, X2, X8 and J1 to human renal epithelial cells 293T, human lung epithelial cells BEAS-2B, rat cardiomyocytes H9C2 and human normal hepatocytes LO2 were evaluated. Finally, the in vivo anti-hypoxia activities of R7, X8 and J1 were further evaluated by atmospheric hypoxia mouse model. The results showed that R7, X8 and J1 exhibited significant anti-hypoxia activities in vitro and in vivo without significant cytotoxicity.