The Varroa destructor (Acari Varroidae) mite is a serious threat to honey bee due to hemolymph feeding and virus transmission. Mite salivary proteins are involved in these interactions. However, the salivary secretome has not been previously characterized. In this paper, the saliva of V. destructor was found to be toxic to the worker larvae of Apis cerana (Hymenoptera Apidae) in the absence of deformed wing virus (DWV) and to stimulate the development of deformed wings in Apis mellifera (Hymenoptera Apidae) adults in the presence of DWV. The salivary secretome was analyzed by nano-liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS). A search of the resulting data against peptide databases using the software Mascot yielded 356, 53, and 9 matched proteins from V. destructor, A. mellifera, and DWV, respectively. The saliva contained Varroa mite proteins identified as important for potential virulence to A. cerana larvae, for the inhibition of harmful microorganisms, for the utilization of bee nutrients, and for antioxidant, oxidation–reduction and detoxification functions as well as A. mellifera proteins identified as nutrients important for mite reproduction. The saliva proteins also contained viral proteins from one virus, DWV. These results provide a strong foundation for understanding the interactions among the Varroa mite, honeybee, and DWV.