As a model bacterial pathogen, Salmonella can gain access into non-phagocytic cells where it proliferates in a unique membrane-bounded compartment.It has long been thought that various nutritional and environmental differences within host cells will force bacterial pathogens to resculpt their proteome.Unfortunately, direct measurements of the in vivo bacterial proteome during infection has been technically challenging, though proteomic analyses of in vitro grown bacteria have been practiced for decades.In order to reveal pathogens adaptation mechanisms while residing in their intracellular niche, here we described a strategy that enables the first comprehensive proteomie survey of Salmonella isolated from infected epithelial cells at distinct stages.Among ~2700 detected bacterial proteins, the abundance levels of >100 proteins were significantly altered at the onsite of Salmonella intracellular replication.The most striking differences are massive induction of bacterial iron-acquisition systems, thereby suggesting severe iron limitation in the host.Consistent with this notion, metabolomic analysis of infected mammalian cells reveals substantially elevated levels of iron-chelating small molecules (known as siderophores) as well.To the best of our knowledge, we are the first to provide the most comprehensive and compelling evidence directly on both the protein and metabolite levels that S.typhimurium encounters severe iron starvation during infection.More importantly, our integrated proteomic and metabolomic strategy described in this study can be readily applied to investigate in principle any intracellular bacterial pathogen, thereby allowing us to gain important insight into their infection biology within the host.