One of the likely consequences of the late Pleistocene megafauna extinction was a reorganization of mammalian communities, including trophic niche shifts of medium- and small-bodied mammals. Traditionally, nitrogen isotope (δ15N) analysis of bulk tissues is used as a proxy for trophic level. However, interpreting temporal or spatial changes in δ15N values is complicated by potential concurrent shifts in (baseline) δ15N values of primary producers. δ15N analysis of individual amino acids (AAs) provides a way to simultaneously track trophic level and baseline δ15N changes. ‘Source’ AAs are routed directly from diet and so their δ15N values do not change with trophic level, whereas ‘trophic’ AAs show a strong relationship between trophic level and δ15N. Here we apply this approach to cotton rat (Sigmodon hispidus) fossils from Hall’s Cave in Texas, a site that spans the last 22,000 years. Cotton rats show a wide range in both carbon (~10‰) and nitrogen (~7‰) isotope values over this interval, but the only significant trend is a decline in δ15N following the late Pleistocene extinction. We found a significant decline in δ15N of source and trophic AAs from the mid to late-Holocene; the offset in δ15N between these groups remained constant This indicates that the decline in bulk S. hispidus values was driven by a shift at the base of the foodweb, rather than a change in S. hispidus trophic position.