Fowl typhoid (FT), caused by Salmonella Gallinarum (SG), inflicts a significant economic burden on the poultry industry. Our study focused on a non-reversible gene deletion to develop a safe and effective SG vaccine strain through lipopolysaccharide (LPS) structural modification. The virulence-attenuated SG JOL914 strain, generated by deleting the global regulator lon gene, was engineered by in-frame deletions targeting the LPS structure. Interruption on LPS deacylation was introduced by pagL deletion, JOL2997, to mitigate endotoxicity induced by the live vaccine. The O-antigen was truncated by rfaL deletion, JOL3004, to compromise virulence and facilitate Differentiating Infected from Vaccinated Animals (DIVA). Both genes were depleted in JOL3016 (ΔlonΔpagLΔrfaL) for their compounding effects. Subcutaneous administration of the engineered strains in chickens showed reduced endotoxicity, particularly demonstrated by JOL3016 with 2.55-fold and 3.79-fold reductions in TNF-α and IL-1β, respectively. Furthermore, discernible effects on body weight gain and the absence of pathological signs demonstrated the safety profiles of the inoculated strains. Administration of mutant strains substantially increased antigen-specific IgY, sIgA, CD4