Pathogenic bacteria have evolved specialized virulence factors (VFs) that enable entry and persistence within host tissues. Here we investigated the in vivo transcriptome of a novel insect pathogenic bacterium, Yersinia entomophaga MH96, to identify key virulence factors involved during different stages of infection (early, middle and late) and at different temperatures (25 and 37 °C) within the hemocoel of larval insect host, Galleria mellonella. Originally, Y. entomophaga was isolated from the cadaver of Costelytra giveni (Coleoptera: Scarabaeidae) larva, which is an endemic and notorious pasture pest of New Zealand. Development of Y. entomophaga as a biopesticide has proven consistent pathogenesis by per os challenge against C. giveni, as well as a wide range of coleopteran, lepidopteran, and orthopteran species. Additionally, a median lethal dose of at least three bacterial cells is sufficient to kill larvae of the greater wax moth G. mellonella within 4 days of injection with similar levels of mortality observed at both 25°C and 37°C. Here we use a novel method to enrich for pathogen transcriptional signal from extracellular Y. entomophaga within the hemolymph of G. mellonella. Sequencing results provided sufficient alignment of transcripts to the reference genome (9 – 91 %) enabling capture of the dynamic range of pathogen gene expression within the host. Striking temperature-dependent regulation was observed for several key VFs, including the insecticidal toxin complex (Yen-TC), type VI secretion system and flagellum, all of which were completely down-regulated at 37 °C compared to 25 °C. These data are driving focused investigations into the temperature-dependent regulation of Yen-TC as well as the characterization of RNA-binding proteins that were shown to be up-regulated during early infection and at 37 °C in the host. This work provides critical insight into the pathobiology and in vivo gene expression of a potentially important biopesticide.