Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the most significant zoonotic pathogens that poses a threat to humans. Previous studies have identified that Salmonella-secreted effector K3 (SseK3) is a novel translated and secreted protein of S. Typhimurium. The objective of this study was to determine whether deletion of the sseK3 gene can attenuate the virulence of S. Typhimurium. To do this, we constructed an sseK3 deletion mutant using the double-exchange allele of the suicide plasmid pRE112ΔsseK3 and assessed the virulence and intracellular proliferation of the mutant. The sseK3 deletion mutant exhibited adhesion and invasion properties similar to those of wild-type (WT) S. Typhimurium, although the virulence and intracellular proliferation of the mutant were significantly reduced compared to that of the WT strain. Furthermore, the observed increase in the median lethal dose (LD(50)) reflects a decrease in the pathogenicity of the sseK3 deletion mutant in a murine model. In summary, we concluded that disruption of sseK3 can attenuate the intracellular proliferation and reduce the virulence of S. Typhimurium.