Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), is one of the most successful infectious diseases in human history and remains a major global health threat with continuing outbreaks in developing countries. Typhoid fever kills 0.2 million and sickens 21 million people every year. Multidrug-resistant strains of S. Typhi is rapidly spreading. One of the most effective countermeasures alternative to antibiotics would be targeting essential virulence mechanisms of typhoid fever. However, little is known about the pathogenic mechanisms specific to S. Typhi. One such essential virulence factor specific to S. Typhi is a recently discovered toxin: typhoid toxin. Typhoid toxin has many unique structural and functional features. This toxin consists of three subunits, CdtB (nuclease), PltA (mono ADP ribosyltransferase), and a homopentamer of PltB (receptor binding), resulting in its unique A2B5 stoichiometry. When administered to laboratory animals, typhoid toxin recapitulated many of the characteristic symptoms of typhoid fever, such as lethargy, malaise, stupor, leukopenia, and neurological complications. It is expected that typhoid toxin plays a similar role during human infection, as supported by clinical reports indicating the abundant presence of anti-typhoid toxin antibodies in the sera of convalescent typhoid patients, as well as our recent studies demonstrating the expression of the specific glycan receptor for typhoid toxin on cells resulting in typhoid symptoms. However, we still do not have a good understanding of how each subunit of typhoid toxin contributes to its virulence. To fill this gap in our knowledge, here we aim to define the precise role(s) of typhoid toxin’s subunits in virulence, with an emphasis on roles of PltA and PltB. The proposed research is significant because this work will offer important insights into the development of effective strategies for targeting the pathogenic mechanisms specific to S. Typhi and attenuating S. Typhi virulence.