Host immune mechanisms were proposed to decline under microgravity conditions during spaceflights, which might result in severe infections in astronauts. Therefore, it was important to investigate the effects of microgravity on infecting organisms and their interaction with host cells. Data showed that simulated microgravity (SMG) conditions markedly increased production of the enterotoxigenic Escherichia coli (ETEC) heat-labile enterotoxin, which induced fluid secretory responses in a mouse model. SMG also enhanced production of tumor necrosis factor-alpha in murine macrophages infected with enteropathogenic E. coli (EPEC). In a similar fashion, simulated microgravity conditions augmented the invasive potential of Salmonella enterica serovar typhimurium and enhanced production of tumor necrosis-factor alpha in S. typhimurium-infected epithelial cells. Furthermore, coculturing of macrophages and S. typhimurium in a simulated microgravity environment resulted in activation of stress-associated mitogen-activated protein kinase kinase 4. Using the antiorthostatic tail suspension mouse model, which simulates some aspects of microgravity, oral inoculation of S. typhimurium markedly reduced the 50% lethal dose compared to mice infected under normal gravitational conditions. Microarray analysis revealed simulated microgravity-induced alterations in the expression of 22 genes in S. typhimurium, and protein expression profiles were altered in both EPEC and S. typhimurium, based on two-dimensional gel electrophoresis. These studies indicated alterations in the virulence potential of bacteria and in host responses to these pathogens under simulated microgravity conditions, which may represent an important environmental signal. Such studies are essential for better understanding bacterial-host cell interactions, particularly in the context of spaceflights and space habitations of long duration.