Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional housekeeping protein secreted by pathogens and involved in adhesion and/or virulence. Previously we reported that enterohemorragic (EHEC) and enteropathogenic (EPEC) Escherichia coli secrete GAPDH into the culture medium. This bacterial protein binds human plasminogen and fibrinogen and remains associated with Caco-2 cells upon infection. In these pathogens, GAPDH secretion is not linked to outer membrane vesicles and depends on growth conditions, although the secretion mechanism is still unknown. EPEC is an attaching and effacing pathogen able to secrete and translocate multiple effector proteins into infected cells through a type III secretion system (T3SS). The secretion process is often dependent on a bacterial chaperone. The chaperone CesT displays broad substrate specificity and plays a central role in the recruitment of multiple type III effectors to the T3SS apparatus. Here we provide genetic evidences on GAPDH secretion through T3SS by EPEC grown in DMEM. Secretion of GAPDH is increased in sepD mutants and abolished in mutants defective in the type III ATPase EscN. Complementation with escN gene restores GAPDH secretion. In addition, we prove by means of pull down experiments, overlay immunoblotting and biolayer interferometry a novel interaction between GAPDH and the chaperone CesT. This interaction, which is strong and slow dissociating, may stabilise a population of GAPDH molecules in a secretion competent-state and target them to the type III secretion apparatus. This is the first description of CesT interaction with a housekeeping protein and its export through T3SS.