Salivary proteins of herbivorous insects play a central role in plant-insect interactions. Spider mite Tetranychus truncatus is a polyphagous agricultural pest harboring various bacterial symbionts. However, whether endosymbionts infection in spider mite alters the host saliva protein remains largely unknown. Here, by using shotgun LC-MS/MS analysis, we identified and characterized the components of saliva in Wolbachia-Spiroplasma infected and uninfected T. truncatus. In total, 177 putative salivary proteins were identified. The function of many proteins remains unknown, while in numerous cases belong to catalytic activity and binding proteins. The saliva enzymes included oxidoreductase, hydratase, isomerase, transferase, protease, esterase, ribonuclease, kinase, lyase and phosphorylase. Other proteins, such as ATP-binding, actin, heat shock proteins and vitellogenin were also detected in the T. truncatus saliva. In addition, we found some of the saliva proteins are mite strain-specific salivary proteins—14 proteins were only found in Wolbachia-Spiroplasma infected spider mite, and 6 proteins were only found in Wolbachia-Spiroplasma uninfected spider mite. Overall, this is the first research to identify and characterize the proteins in saliva of facultative symbionts-infected and uninfected spider mites, T. truncatus. Our novel findings revealed that the presence of bacterial symbionts affected the saliva components of spider mites, opening the path for future studies.