This study was aimed to observe the effects of vitamin B, vitamin B, and niacin supplementation on hepatic gene expression profiles in mice exposed to acute hypoxia. Thirty mice were randomly divided into normal, acute hypoxia, and acute hypoxia plus vitamin B, vitamin B, and niacin supplementation groups and fed corresponding diets for 2 weeks and then exposed to a simulated altitude of 6000 m for 8 h. Hepatic gene expression profiles were analyzed using a microarray technique. Several biochemical markers were also assayed. The results showed that a total of 2476 genes were expressed differentially after acute hypoxia exposure (1508 upregulated genes and 968 downregulated genes). Compared with the acute hypoxia group, there were 1382 genes differentially expressed (626 upregulated genes and 756 downregulated genes) in the acute hypoxia plus vitamin B, vitamin B, and niacin supplementation group. Pathway analysis indicated that carbohydrate, lipid, and amino acid metabolism, as well as electron transfer chain, were improved to some extent after vitamin B, vitamin B, and niacin supplementation. Supportive results were obtained from biochemical assays. Our findings suggest that the supplementation of vitamin B, vitamin B, and niacin is beneficial in improving nutritional metabolism partly via gene expression under acute hypoxia condition.