Edwardsiella tarda is a Gram-negative, facultative aerobic pathogen which infects multifarious hosts including fish, amphibians and human beings. A twin-arginine translocation (Tat) gene cluster important for high-salt tolerance in E. tarda was identified previously. Here the genetic structure and pleiotropic roles of the Tat system in physiological adaptation of the bacterium were further characterized. Functional analysis indicated that tatD was not required for Tat export process and tatE might be an allelic gene of tatA in the bacterium. The results showed that disruption in the Tat system did not affect the morphology and biofilm formation in E. tarda, but did affect motility, hemagglutination, cell aggregation and infection of eukaryotic cells (e.g. macrophage J774a). Comparative proteomics analysis of subcellular proteins using two-dimensional gel electrophoresis and a qualitative shotgun protein sequencing method were implemented to identify proteins differentially expressed in E. tarda EIB202 vs. ∆tatABCD. The results revealed a large repertoire of differentially expressed proteins (n = 61), shedding light on the Tat system associated with virulence and stress-associated processes in E. tarda.