[Objectives]  To investigate the physiological responses of Apis mellifera worker pupae to Varroa destructor, and illustrate the effects of this parasite on the development of honey bees. [Methods]  A gelatin capsule-based Varroa in vitro rearing system was used to monitor the development of honey bee pupae infested by Varroa mites. Capsules containing early fifth instar worker larvae were inoculated with 0 (Control), 1 and 2 mites, respectively, and kept at an appropriate temperature in an incubator until the bees emerged. The appearance, body weight and mortality of larvae, and the DWV level of both hosts and parasites, were recorded and analyzed three (D3), six (D6) and nine days (D9) after Varroa inoculation and at the time of emergence. [Results]  A range of deformities such as crippled or deformed wings, a shortened abdomen, developmental retardation indicated by delayed compound eye pigmentation, were observed and the deformity rate was significantly correlated with the number of mites (P < 0.000 1). The body weight of Varroa-infested bees was significantly less than that of the control group on D6, D9, and at emergence (P < 0.001), and was negatively correlated with the number of mites (P < 0.000 1). Bee’s life spans were inversely correlated with the number of mites (P < 0.001). Relative DWV levels were significantly up-regulated by Varroa parasitism on D9, and were positively correlated with the number of mites (P < 0.001). In addition, expressed DWV RNAs were positively correlated between hosts and the parasites (P < 0.001). [Conclusion]  Varroa parasitism both retards the development and increases the mortality of honey bee worker pupae, thereby posing a significant threat to colony health.