Food limitation is the most common environmental challenge faced by animals and the capacity of animals to survive prolonged periods of starvation is linked to their diet and nutritional status. The objective of this study is to investigate the effects of nutrition on starvation resistance in Drosophila melanogaster. Experimental flies were given ad libitum access to artificial diets differing in concentrations and ratios of protein and carbohydrate for 5 days before they were assayed for starvation time, body composition and life-history parameters. Starvation resistance in Drosophila was greatly influenced by the dietary protein:carbohydrate (P:C) ratio, but neither by the caloric content of the diet nor by dietary carbohydrate alone. Starvation resistance was strongest at the lowest P:C ratio and declined with rising P:C ratio. While starving, Drosophila underwent a dramatic transition in the utilization of physiological fuels, switching from the early phase characterized by preferential consumption of non-lipid substrates to the next phase in which they began to mobilize lipids as fuels for enduring starvation. Our results highlight the importance of nutrition as a key factor determining starvation responses of Drosophila.