Abstract. Root litter decomposition is a major component of carbon (C) cycling in grasslands, where it provides energy and nutrients for soil microbes and fauna. This is especially important in grasslands where fire is common and removes aboveground litter accumulation. In this study, we investigated whether fire affects root decomposition and C flow through the belowground food web. In a greenhouse experiment, we applied ¹³C-enriched big bluestem (Andropogon gerardii) root litter to intact tallgrass prairie soil cores collected from annually burned (AB) and infrequently burned (IB) treatments at the Konza Prairie Long Term Ecological Research (LTER) site. Incorporation of ¹³C into microbial phospholipid fatty acids and nematode trophic groups was measured on six occasions during a 180-day decomposition study to determine how C was translocated through the soil food web. Results showed significantly different soil communities between treatments and higher microbial abundance for IB. Root decomposition occurred rapidly and was significantly greater for AB. Microbes and their nematode consumers immediately assimilated root litter C in both treatments. Root litter C was preferentially incorporated in a few groups of microbes and nematodes, but depended on burn treatment: fungi, Gram-negative bacteria, Gram-positive bacteria, and fungivore nematodes for AB and only omnivore nematodes for IB. The overall microbial pool of root-litter-derived C significantly increased over time but was not significantly different between burn treatments. The nematode pool of root-litter-derived C also significantly increased over time, and was significantly higher for the AB treatment at 35 and 90 days after litter addition. In conclusion, the C flow from root litter to microbes to nematodes is not only measurable but also significant, indicating that higher nematode trophic levels are critical components of C flow during root decomposition, which, in turn, is significantly affected by fire. Not only does fire affect the soil community and root decomposition, but the lower microbial abundance, greater root turnover, and the increased incorporation of root litter C by microbes and nematodes for AB suggests that annual burning increases root-litter-derived C flow through the soil food web of the tallgrass prairie.