The mite Varroa destructor is currently the greatest threat to apiculture as it is causing a global decrease in honey bee colonies. However, it rarely causes serious damage to its native hosts, the eastern honey bees Apis cerana. To better understand the mechanism of resistance of A. cerana against the V. destructor mite, we profiled the metabolic changes that occur in the honey bee brain during V. destructor infestation. Brain samples were collected from infested and control honey bees and then measured using an untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based global metabolomics method, in which 7918 and 7462 ions in ESI+ and ESI- mode, respectively, were successfully identified. Multivariate statistical analyses were applied, and 64 dysregulated metabolites, including fatty acids, amino acids, carboxylic acid, and phospholipids, amongst others, were identified. Pathway analysis further revealed that linoleic acid metabolism; propanoate metabolism; and glycine, serine, and threonine metabolism were acutely perturbed. The data obtained in this study offer insight into the defense mechanisms of A. cerana against V. destructor mites and provide a better method for understanding the synergistic effects of parasitism on honey bee colonies.