Rationale: The trophic enrichment factor (TEF) is a parameter reflecting the difference in isotopic ratio between a consumer's tissues and diet, used in isotopic ecology and paleoecology to track dietary habits. The TEF of sulfur is believed to be low, but was, until now, only documented in a limited number of taxa. In this study we use a subfossil accumulation of bones from a red fox (Vulpes vulpes) den to verify the TEF for sulfur in fox bone collagen. Methods: Collagen was extracted from 30 samples of subfossil bones, including foxes and their prey. The δ S values of the bone collagen samples were measured with an elemental analyzer connected to an isotope ratio mass spectrometer. The TEF was calculated as [Δ S = (mean δ S in predator) - (mean δ S in prey)], using taphonomic indices to estimate the mean diet, and calculated separately for different age classes of the predator.Results: We modeled 12 variants of TEF for different estimations of the diet composition and for three fox age classes (adult, subadult, and juvenile). The estimated TEF values range from -0.54 to +0.03‰ and are similar to TEFs known for other mammals. Absolute TEF values are nearly equal to or lower than the analytical error, which is ±0.4‰.Conclusions: For the first time, we present direct δ S data for the bone collagen of a free-living predator and its naturally selected prey. Our results indicate very low or even slightly negative TEF values for sulfur. Furthermore, according to our results, the δ S value should not be considered a reliable indicator of trophic position in terrestrial food webs but rather, it should be used to disentangle different food webs based on different primary producers.