Simple SummaryThis study explored the interaction effects of high temperature stress andWolbachiainfection on the biological characteristic of fruit flyDrosophila melanogaster. Results showed that high temperature andWolbachiainfection had a two-way interaction effect on hatching rate, developmental durations, emergence rate, body weight and body length of F1, F2and F3flies, and the interaction effect also existed on oviposition amount of F3flies, and on pupation rate of F2and F3flies. The study also showed that high temperature stress deceased theWolbachiavertical transmission efficiency between generations, and both high temperature stress andWolbachiainfection can reduce the survival ofD. melanogaster. The results indicated that high temperature stress andWolbachiainfection had negative effects on the growth and development ofD. melanogaster.AbstractIt was reported that temperature affects the distribution ofWolbachiain the host, but only a few papers reported the effect of the interaction between high temperature andWolbachiaon the biological characteristic of the host. Here, we set four treatmentDrosophila melanogastergroups:Wolbachia-infected flies in 25 °C (W+M),Wolbachia-infected flies in 31 °C (W+H),Wolbachia-uninfected flies in 25 °C (W-M),Wolbachia-uninfected flies in 31 °C (W-H), and detected the interaction effect of temperature andWolbachiainfection on the biological characteristic ofD. melanogasterin F1, F2and F3generations. We found that both temperature andWolbachiainfection had significant effects on the development and survival rate ofD. melanogaster. High temperature andWolbachiainfection had interaction effect on hatching rate, developmental durations, emergence rate, body weight and body length of F1, F2and F3flies, and the interaction effect also existed on oviposition amount of F3flies, and on pupation rate of F2and F3flies. High temperature stress reduced theWolbachiavertical transmission efficiency between generations. These results indicated that high temperature stress andWolbachiainfection had negative effects on the morphological development ofD. melanogaster.