Objective Mouse infection models are frequently used to study the host-pathogen interaction studies. However, due to several constraints, there is an urgent need for a simple, rapid, easy to handle, inexpensive, and ethically acceptable in vivo model system for studying the virulence of enteropathogens. Thus, the present study was performed to develop the larvae of Helicoverpa armigera as a rapid-inexpensive in vivo model system to evaluate the effect of Yersinia enterocolitica strain 8081 on its midgut via a label-free proteomic approach. Results Helicoverpa armigera larvae fed with Yersinia enterocolitica strain 8081 manifested significant reduction in body weight and damage in midgut. On performing label-free proteomic study, secretory systems, putative hemolysin, and two-component system emerged as the main pathogenic proteins. Further, proteome comparison between control and Yersinia added diet-fed (YADF) insects revealed altered cytoskeletal proteins in response to increased melanization (via a prophenoloxidase cascade) and free radical generation. In concurrence, FTIR-spectroscopy, and histopathological and biochemical analysis confirmed gut damage in YADF insects. Finally, the proteome data suggests that the mechanism of infection and the host response in Y. enterocolitica-H. armigera system mimics Yersinia-mammalian gut interactions. Conclusions All data from current study collectively suggest that H. armigera larva can be considered as a potential in vivo model system for studying the enteropathogenic infection by Y. enterocolitica strain 8081. Graphic abstract