The fructose analogue 2,5-anhydro-D-mannitol (2,5-AM) stimulates feeding behavior in rats apparently through its effects on hepatic energy metabolism, where it reduces glucose utilization, traps phosphate, and decreases ATP. The extent to which the magnitude and duration of the eating response are dependent on the ability of the liver to switch to fat oxidation for energy production was investigated by manipulating substrate availability through dietary and pharmacological means. Rats adapted to a high-fat, low-carbohydrate diet preferentially use fat fuels for hepatic energy production and were insensitive to the effects of 2,5-AM on food intake. The lack of an eating response occurred despite similar changes in plasma fuels and liver glycogen compared with rats fed a low-fat, high-carbohydrate diet. In contrast, inhibiting fatty acid oxidation with methyl palmoxirate, which blocks transport of long-chain fatty acids to the mitochondria, potentiated the ability of 2,5-AM to stimulate feeding without altering its effects on plasma and liver fuels. These data demonstrate that the eating response to 2,5-AM is modulated by the availability of fat fuels and implicate a mechanism for initiation of feeding that is not dependent on inhibition of carbohydrate metabolism per se but rather integrates information about the use of both types of fuels.