Yersinia ruckeri is a Gram negative bacteria causing yersiniosis in freshwater and marine fish. Lipid A, important for pathogenesis of Gram negative bacteria, biosynthesis pathway requires nine enzyme catalyzed steps. Although there are nine genes encoding lipid A biosynthesis in bacteria, biosynthesis of lipopolysaccharides relies on lpxD gene that encodes the third pathway enzyme. The roles of LpxD in Y. ruckeri virulence have not been studied. In the present study, in-frameshift deletion of lpxD gene and their role in Y ruckeri virulence in rainbow trout were determined. For this purpose, 92% of the Y. rucked lpxD genes were deleted by homologous recombination. After running in SDS-PAGE and staining with silver stain, no LPS was detectable in the Y. ruckeri Delta lpxD mutant. Virulence and immunogenicity of the Y. ruckeri Delta lpxD mutant (Yr Delta lpxD) were determined in rainbow trout. Rainbow trout immunized with Yr Delta lpxD with immersion, or intraperitoneal injection method displayed superior protection (relative percentage survival >= 84%) after exposure to wild type Y. ruckeri. In conclusion, our results indicated that deletion of the IpxD gene causes significant attenuation of Y ruckeri in rainbow trout, and LPS deficient Yr Delta lPxD could be used as a live attenuated vaccine against Y. rucked in rainbow trout. This vaccine can protect fish and it can be applied to fish with different methods such as immersion or injection. (C) 2016 Elsevier Ltd. All rights reserved.