Salmonella infection is a major public health concern worldwide, and causes huge mortality and morbidity especially in developing countries. Even though there are significant information about Salmonella pathogenesis and whole genome sequencing also done, but functional characterizations of certain genes are still unexplored. The rspA (STM14_1818) is one such gene with putative dehydratase function, and its role in pathogenesis is unknown. In our study we found that the rspA gene of S. Typhimurium (WT-STM) is upregulated in the MGM-MES media in a time-dependent manner. Thus, we generated the rspA knockout strain (&DeltarspA-STM) and complement strain (&DeltarspAc-STM) in S. Typhimurium 14028. The &DeltarspA-STM was sensitive to higher ROS concentrations and resistant to various antibiotics, complement system and cationic antimicrobial peptides (CAMP). In LB medium, the SP1-1 genes responsible for invasion, SPI-II genes, biofilm related genes and other stress related genes are downregulated in &DeltarspA-STM.