In Salmonella enterica serovar Typhimurium, many genes encoded within Salmonella pathogenicity island 1 (SPI1) are required to induce intestinal/diarrheal disease. In this study, we compared the expression of four SPI1 genes (hilA, invF, prgH, and sipC) under shaking and standing culture conditions and found that the expression of these genes was highest during the transition from the exponential to stationary phase under shaking conditions. To identify regulators associated with the stationary phase-dependent activation of SPI1, the effects of selected regulatory genes, including relA/spoT (ppGpp), luxS, ihfB, hfq, and arcA, on the expression of hilA and invF were compared under shaking conditions. Mutations in the hfq and arcA genes caused a reduction in hilA and invF expression (more than 2-fold) in the early stationary phase only, whereas the lack of ppGpp and IHF decreased hilA and invF gene expression during the entire stationary phase. We also found that hfq and arcA mutations caused a reduction of hilD expression upon entry into the stationary phase under shaking culture conditions. Taken together, these results suggest that Hfq and ArcA regulate the hilD promoter, causing an accumulation of HilD, which can trigger a stationary phase-dependent activation of SPI1 genes under shaking culture conditions.