Free-living amoebae act as environmental hosts of several intracellular pathogens. We examined the interaction between Acanthamoeba rhysodes and Salmonella, a human intracellular pathogen. There was no difference among three different serovars of Salmonella in terms of their growth within A. rhysodes over time. The number of intracellular bacteria increased at 6 h post-infection, and the viability of A. rhysodes was significantly reduced at 24 h post-infection. Amoebic cell death was characterized by TUNEL and Annexin V assay, without DNA ladder identified, indicating an apoptosis-like cell death in Salmonella-infected A. rhysodes. Global gene expression screening between intracellular and extracellular Salmonella by microarray and quantitative PCR showed that genes from Salmonella pathogenicity islands and virulence plasmid were up-regulated within A. rhysodes. The phase-dependent expression pattern suggests their distinct roles in the pathogenesis. A. rhysodes and Salmonella provide a model to study transient symbiosis between bacterial pathogens and protozoa in an aquatic ecosystem.