By combining intravital multiphoton microscopy and bacterial genetics we have developed a technique enabling real‐time imaging of bacterial proliferation and tissue responses in a live animal. Spatial and temporal control of the infection process was achieved by microinjecting GFP⁺‐expressing uropathogenic Escherichia coli (UPEC) into tubules of exteriorized kidneys in live rats. GFP⁺ was introduced in the clinical UPEC strain CFT073 as a single‐copy chromosomal gene fusion. Within hours, bacterial colonization was accompanied by marked ischaemic effects, perivascular leakage, loss of tubular integrity and localized recruitment of immune cells. The pathophysiology was altered in response to an isogenic bacterial strain lacking the exotoxin haemolysin, revealing the subtle and temporal roles of bacterial virulence factors in vivo. Microdissection and RNA extraction of the injected nephron allowed molecular analysis of prokaryotic and eukaryotic gene expression. The techniques described here can be applied to study the integrated cell communication evoked by a variety of bacterial pathogens, assisting in the design of strategies to combat bacterial infections.