Fungus-growing termites of the genus Macrotermes cultivate symbiotic fungi (Termitomyces) in their underground nest chambers to degrade plant matter collected from the environment. Although the general mechanism of food processing is relatively well-known, it has remained unclear whether the termites get their nutrition primarily from the fungal mycelium or from plant tissues partly decomposed by the fungus. To elucidate the flows of carbon and nitrogen in the complicated food-chains within the nests of fungus-growing termites, we determined the stable isotope signatures of different materials sampled from four Macrotermes colonies in southern Kenya. Stable isotopes of carbon revealed that the termite queen and the young larvae are largely sustained by the fungal mycelium. Conversely, all adult workers and soldiers seem to feed predominantly on plant and/or fungus comb material, demonstrating that the fungal symbiont plays a different nutritional role for different termite castes. Nitrogen stable isotopes indicated additional differences between castes and revealed intriguing patterns in colony nitrogen cycling. Nitrogen is effectively recycled within the colonies, but also a presently unspecified nitrogen source, most likely symbiotic nitrogen-fixing bacteria, seems to contribute to nitrogen supply. Our results indicate that the gut microbiota of the termite queen might be largely responsible for the proposed nitrogen fixation.