Aims: The aim of this study was to investigate the role of membrane-bound lytic murein transglycosylase A (MltA) in a bacterial fish pathogen Edwardsiella tarda. Methods and Results: An mltA in-frame deletion mutant (?mltA) and an mltA overexpression strain (mltA+) of Edw.similar to tarda were constructed through double-crossover allelic exchange and by transformation of a low-copy plasmid carrying the intact mltA into the ?mltA mutant, respectively. Either inactivation or overexpression of MltA in Edw.similar to tarda resulted in elevated sensitivity to beta-lactam antibiotics and lower viability in oligotrophic or high osmotic environment than wild-type strain. Autolysis induced by EDTA was reduced in ?mltA strain, while mltA+ strain was virtually flimsy, indicating that MltA is responsible for the lysis effect. Moreover, mltA+ strain exhibited significant increases in lipopolysaccharide (LPS) biosynthesis and virulence to zebra fish compared with wild-type strain. Conclusions: The results indicated that MltA plays essential roles in beta-lactam antibiotics and environmental stresses resistance, autolysis, LPS biosynthesis and pathogenicity of Edw.similar to tarda. This is the first report that MltA has a virulence-related function in Edw.similar to tarda. Significance and Impact of the Study: This study provided useful information for further studies on pathogenesis of Edw.similar to tarda.