Hypoxia preconditioning (HPC) is associated with many complicated pathophysiological and biochemical processes that integrated and regulated via molecular levels.HPC could protect cells, tissues, organs and systems from hypoxia injury, but up to date, the molecular mechanism still remained unclear.The acute and repetitive hypoxia preconditioning model was constructed and the related parameters were observed.The highthroughput microarray analysis and multiple bioinformatics were used to explore the differentially expressed genes in HPC mice brain and the related gene network, pathways and biological processes related to HPC.The 2DDIGE coupled with MALDITOF/TOFMS was performed to identify these proteins that were differentially expressed during HPC.The UPLCHRMS based metabolomics method was utilized to explore the key endogenous metabolites and metabolic pathways related to HPC.The results showed that (1) 1175 differentially expressed genes in HPC mice brain were identified.Fourteen of these genes were the related hub genes for HPC, including Cacna2d1, Grin2a, Npy1r,Mef2c, Epha4, Rxfp1, Chrm3, Pde1a, Atp2b4, Glra1, Idi4, Fgf1, Grin2b and Cda.The change trends of all the detected genes by RTPCR were consistent with the data of gene chips.There were 113 significant functions upregulated and 138 significant functions downregulated in HPC mice.(2) About 2100 proteins were revealed via the gel imaging and spot detection.66, 45 and 70 of proteins were found to have significantly difference between the control group and three times of HPC group, the control and six times of HPC, and the three times of HPC and six times of HPC group.(3) Some endogenous metabolites such as phenylalanine, valine, proline, leucine and glutamine were increased, while creatine was decreased, both in HPC brain and heart; in addition, γaminobutyric acid was markedly decreased in brain.The sphingolipid metabolic pathways were noticed due to the low pvalue and high pathway impact.Especially, the sphingolipid compound sphingomyelin, ceramide, glucosylceramide,galactosylceramide and lactosylceramide were mapping in this metabolic pathway.Interestingly, these sphingolipid metabolites with olefinic bond in the long fatty chain were upregulated, while those sphingolipids without olefinic bond were downregulated.The functions of these differentially expressed genes mainly involved the cellular processes including MAPK pathway, ion transport, neurotransmitter transport and neuropeptide signal pathway.The protein levels related the ATP synthesis and citric acid cycle decreased while the proteins with the glycolysis and oxygenbinding increased.Glutathione, GNBP1 and GPD1L were related to preventing hypoxic damage.The results indicated that C24 : 1Cers played a critical role in HPC and had potential in endogenous protective mechanism.The combinations of the system omics data of the different molecules were sufficient to give a further understanding of the molecular pathways affected by HPC.Our data provided an important insight to reveal the protection mechanism of HPC.