Background/Question/Methods Larval stages of mosquitoes are subject to several environmental stressors that can alter their phenotypic traits and immune responses to viral pathogen infection. The application of pesticides in mosquito control programs can occur under a variety of environmental temperatures where there is a risk of viral pathogen transmission. Here, we examined whether phenotypic traits and susceptibility to Zika virus (ZIKV) infection of Aedes aegypti can be altered by the interactions between pyriproxyfen and different temperatures (20°C and 30°C) during larval stages. Results/Conclusions Larval development time was lengthened at 20°C and in the presence of pyriproxyfen. Pupal-adult metamorphosis inhibition by pyriproxyfen was higher at elevated temperature than low temperature. Wing length of females was longer at 20°C and shorter at 30°C. Infection, disseminated infection, and saliva infection of ZIKV in females were enhanced by pyriproxyfen at both 20°C and 30°C. Zika virus titers in female tissues were not affected by pyriproxyfen or temperatures. Our results revealed that the interactive effects between larval environmental stressors can significantly influence mosquito phenotypic traits and susceptibility to viral pathogen infection. The pyriproxyfen-induced enhancement of ZIKV infection in females should be a consideration when implementing pyriproxyfen in mosquito control programs.