Objective To explore metabolic changes after acute hypoxia and modulating effect of vitamins B1,B2, and PP supplementation in mice exposed to acute hypoxia. Methods Fifty male Kunming mice were randomly divided into 5 groups: normal, acute hypoxia, acute hypoxia with 2, 4 and 8 time- vitamins B1, B2, and PP supplementation. All mice were fed with corresponding diets for two weeks and then were exposed to a simulated altitude of 6 000 meters for 8 h, except for the normal group. Nuclear magnetic resonance analysis was used to identify the changes of serum metabolic profiles. Results There were significant changes in some serum metabolites under induced acute hypoxia, essentially relative increase in the concentrations of lactate, sugar and lipids and decrease in ethanol. The serum levels of choline, succinate, taurine, alanine, and glutamine also increased and phosphocholine decreased in the acute hypoxia group. After vitamins B1, B2, and PP supplementation, all these metabolic changes gradually recovered. Conclusion Significant changes in serum metabolic profile were observed by metabolomics in mice exposed to acute hypoxia, and vitamins B1, B2, and PP supplementation proved to be beneficial to improving some metabolic pathways. It is suggested that the dietary intakes of vitamins B1, B2, and PP should be increased under hypoxia condition.