Transportation stress is one of the most serious issues in the management of yak. Previous studies have demonstrated that transport stress is caused by a pro-oxidant state in the animal resulting from an imbalance between pro-oxidant and antioxidant status. In this context, vitamin C has the ability to regulate reactive oxygen species (ROS) synthesis and alleviate oxidative stress. Although this effect of vitamin C is useful in pigs, goats and cattle, the effect of vitamin C on the mitigation of transport stress in yaks is still unclear. The purpose of this study was to better assess the metabolic changes induced by the action of vitamin C in yaks under transportation stress, and whether these changes can influence antioxidant status. After the yaks arrived at the farm, control or baseline blood samples were collected immediately through the jugular vein (VC_CON). Then, 100 mg/kg VC was injected intramuscularly, and blood samples were collected on the 10th day before feeding in the morning (VC). Relative to the control group, the VC injection group had higher levels of VC. Compared with VC_CON, VC injection significantly (P< 0.05) decreased the blood concentrations of ALT, AST, T-Bil, D-Bil, IDBIL, UREA, CRP and LDH. However, VC injection led to greater (P< 0.05) AST/ALT and CREA-S relative to VC_CON. There was no difference (P> 0.05) in GGT, ALP, TBA, TP, ALBII, GLO, A/G, TC, TG, HDL-C, LDL-C, GLU andl-lactate between VC_CON and VC. The injection of VC led to greater (P< 0.05) concentration of MDA, but did not alter (P> 0.05) the serum concentrations of LPO and ROS. The injection of VC led to greater (P< 0.05) serum concentrations of POD, CAT and GSH-PX. In contrast, lower (P< 0.05) serum concentrations of SOD, POD and TPX were observed in VC relative to VC_CON. No difference (P> 0.05) in GSH, GSH-ST and GR was observed between VC_CON and VC. Compared with the control group, metabolomics using liquid chromatography tandem–mass spectrometry identified 156 differential metabolites withP< 0.05 and a variable importance in projection (VIP) score > 1.5 in the VC injection group. The injection of VC resulted in significant changes to the intracellular amino acid metabolism of glutathione, glutamate, cysteine, methionine, glycine, phenylalanine, tyrosine, tryptophan, alanine and aspartate. Overall, our study indicated that VC injections were able to modulate antioxidant levels by affecting metabolism to resist oxidative stress generated during transport.