In this study, we present the first thorough trophic characterization of cold seep macrofaunal communities on the Gulf of Mexico lower continental slope (> 1000 m depth). We analyzed tissue delta C-13, delta N-15, and delta S-34 of vestimentiferan tubeworms, bathymodiolin mussels, vesicomyid clams, and their associated macrofaunal communities from discrete collections made across the entire lower slope. Over half of macrofauna associated with mussels and about half associated with vestimentiferans had delta C-13 values below -45%. We also observed high spatial variability in the delta C-13 values of entire local communities, and the delta C-13 of associated fauna were significantly correlated with the delta C-13 compositions of the symbiotic species from the same location. These data indicate widespread incorporation of methane-derived carbon in mussel and vestimentiferan communities. This finding was particularly surprising in communities associated with older vestimentiferans, given the low rates of seepage observed in similar communities on the upper slope. On average, delta N-13 values in mussels and their associates were significantly more depleted and more variable than vestimentiferans, clams, and their associates, and there was a significant linear relationship between tissue delta N-15 values of mussels and their associated communities. The tissue delta S-34 values in macrofauna associated with vestimentiferans were more variable and significantly more depleted than mussel associates (delta S-34 = -16.8 to + 19.1% for vestimentiferan associates and delta S-34 = -3.1 to + 20.8% for mussel associates), consistent with higher isotopic fractionation during sulfate reduction in vestimentiferan habitats and a potentially higher nutritional contribution of sulfide-derived organic sulfur in vestimentiferan communities.