The honey bee is a significant crop pollinator and key model insect for understanding social behavior, disease transmission, and development. The ectoparasitic Varroa destructor mite put threats on the honey bee industry. A Varroa toxic protein (VTP) from the saliva of Varroa mites contributes to the toxicity toward Apis cerana and the deformed wing virus elevation in Apis mellifera. However, the immune response and hemolymph microbiota of honey bee species after the injection of recombinant VTP has not yet been reported. In this study, both A. cerana and A. mellifera worker larvae were injected with the recombinant VTP. Then the expressions of the honey bee immune genes abaecin, defensin, and domeless at three time points were determined by qRT–PCR, and hemolymph microbial community were analyzed by culture-dependent method, after recombinant VTP injection. The mortality rates of A. cerana larvae were much higher than those of A. mellifera larvae after VTP challenge. VTP injection induced the upregulation of defensin gene expression in A. mellifera larvae, and higher levels of abaecin and domeless mRNAs response in A. cerana larvae, compared with the control (without any injection). Phosphate buffer saline (PBS) injection also upregulated the expression levels of abaecin, defensin, and domeless in A. mellifera and A. cerana larvae. Three bacterial species (Enterococcus faecalis, Staphylococcus cohnii, and Bacillus cereus) were isolated from the hemolymph of A. cerana larvae after VTP injection and at 48 h after PBS injections. Two bacterial species (Stenotrophomonas maltophilia and Staphylococcus aureus) were isolated from A. mellifera larvae after VTP challenge. No bacterial colonies were detected from the larval hemolymph of both honey bee species treated by injection only and the control. The result indicates that abaecin, defensin, and domeless genes and hemolymph microbiota respond to the VTP challenge. VTP injection might induce the dramatic growth of different bacterial species in the hemolymph of the injected larvae of A. mellifera and A. cerana, which provide cues for further studying the interactions among the honey bee, VTP, and hemolymph bacteria.