Laboratory studies have shown that high levels of dietary unsaturated fatty acids prolong torpor and lower body temperatures in hibernating herbivorous rodents, which may in turn improve winter survival. The importance of nutritional ecology in relation to hibernation in insectivorous hibernators is unknown. We therefore studied fatty acid composition of dietary insects and the depot fat of echidnas Tachyglossus aculeatus (Monotremata) during the pre-hibernation season and compared depot fat fatty acid composition before and after hibernation. Echidna depot fat fatty acid composition during the pre-hibernation season was almost identical to that of the most abundant prey species, the ant Iridomyrmex sp. Oleic acid (C18:1) was by far the most common fatty acid in both Iridomyrmex sp. (60%) and echidna depot fat (62%). After about 5 months of hibernation and an 18% loss of body mass, echidna fatty acid composition had changed significantly. The percentage of the monounsaturated oleic acid (C18:1) and palmitoleic acid (C16:1) had declined, whereas that of the saturated fatty acids (C12:0, C16:0, C18:0) and the polyunsaturated linoleic acid (C18:2) had increased. Our study suggests that, unlike herbivorous rodent hibernators, echidnas rely to a large extent on monounsaturated fatty acids as fuel for hibernation, reflecting the most common fatty acid in their food. Moreover, it appears that the high concentration of monounsaturated fatty acids compensates for the moderate availability of polyunsaturates and enables them to hibernate at low body temperatures.