To evaluate the effects of microgravity on virulence genes in Salmonella, we studied the ability of various Salmonella deletion mutants to kill wild-type Caenorhabditis elegans nematodes at the larval and adult stages. Simultaneous studies were performed utilizing spaceflight, clinorotation, and static ground controls. Nematodes, Salmonella, and growth media were separated until exposed to true or simulated microgravity, and then mixed and grown for 48h. Experiments were terminated by paraformaldehyde fixation, and optical density measurements were used to assay residual microorganisms. Prior flight in space led to reduced virulence of wild-type Salmonella when subsequently evaluated in a ground-based virulence assay with carefully matched inocula for never-flown Salmonella controls. However, when the virulence assay was conducted in spaceflight, there was only a minimal change in the virulence of wild-type Salmonella toward C. elegans. Deletion of pipA, a gene in Salmonella pathogenicity island-5, reduced Salmonella virulence toward wild-type and Tol1-deletion L2 larvae in spaceflight but had no effect on virulence for Tol1-deletion adult worms in spaceflight. PipA-deletion Salmonella were also less virulent toward wild-type L2 larvae in clinorotation, but showed a paradoxical increased virulence toward Tol1-deletion L2 larvae in clinorotation.