Dd-TRAP1 is a Dictyostelium homologue of tumor necrosis factor receptor-associated protein 1 (TRAP-1). Dd-TRAP1 is located in the cortex of cells growing at a low density, but was found to be translocated to mitochondria with the help of a novel prestarvation factor that was accumulated in growth medium along with increased cell densities. The knockdown mutant of Dd-TRAP1 (TRAP1-RNAi cells) exhibited a significant defect in prestarvation response. Although TRAP1-RNAi cells showed normal expressions of classical prestarvation genes [dscA (discoidin I) and car1 (carA; cAMP receptor)], the expression of differentiation-associated genes (dia1 and dia3) induced by the prestarvation response were markedly repressed. By contrast, transformants overexpressing Dd-TRAP1 showed an early prestarvation response and also increased expression of dia1 and dia3 in a cell-density-dependent manner. Importantly, introduction of Dd-TRAP1 antibody into D. discoideum Ax-2 cells by electroporation inhibited the translocation of Dd-TRAP1 from the cortex to mitochondria and greatly inhibited the initiation of differentiation. Taken together, these results indicate that Dd-TRAP1 is translocated to mitochondria by sensing the cell density in growth medium and enhances the early developmental program through a novel prestarvation response.