Australia has the highest reported incidence of salmonellosis in the developed world, with three times higher reported cases compared to the UK or US. Recently, there has been a rapid emergence of a monophasic variant of Salmonella enterica serovar Typhimurium, 4,[5],12:i:-. These monophasic strains are characterised by deletions in the FljAB operon, preventing expression of the phase two flagellar antigen FljB, and the acquisition of multidrug resistance factors. As the current published work on S. Typhimurium monophasic variants has primarily focussed on genomic and multidrug resistance studies, we were interested in investigating their growth characteristics and host cellular responses in vitro. We infected human macrophages and colonic epithelial cells with a panel of monophasic S. Typhimurium strains and compared their intracellular growth and effect on cell viability with a type strain (SL1344) and circulating biphasic isolates. We observed limited infection of monophasic strains in epithelial cells with the exception of two strains which were highly attenuated. However, in macrophages we observed 5-10-fold higher intracellular replication of most monophasic strains compared to SL1344, despite lower initial infection rates. Intriguingly, this did not adversely affect host cell viability, which remained equal to or higher than SL1344-infected cells across all monophasic strains. We also show decreased activation of caspases 1, 3 and 8 at early stages of monophasic S. Typhimurium replication compared to SL1344, suggesting that the monophasic strains were able to replicate to high levels within macrophages without activating the intracellular cell death pathways typically observed in S. Typhimurium infections. This could in turn facilitate systemic spread of the pathogen through the host, and has implications for the recent emergence of these monophasic isolates in Australia. Our current research aims to determine the mechanism by which monophasic S.Typhimurium strains can modulate host intracellular detection and immune responses in macrophages.