The sea otter (Enhydra lutris) is a marine mammal hunted to near extinction during the 1800s. Despite their well-known modern importance as a keystone species, we know little about historical sea otter ecology. Here, we characterize the ecological niche of ancient southern sea otters (E. lutris nereis) using δ13C analysis and δ15N analysis of bones recovered from archaeological sites spanning ~7,000 to 350 years before present (N = 112 individuals) at five regions along the coast of California. These data are compared with previously published data on modern animals (N = 165) and potential modern prey items. In addition, we analyze the δ15N of individual amino acids for 23 individuals to test for differences in sea otter trophic ecology through time. After correcting for tissue-specific and temporal isotopic effects, we employ nonparametric statistics and Bayesian niche models to quantify differences among ancient and modern animals. We find ancient otters occupied a larger isotopic niche than nearly all modern localities; likely reflecting broader habitat and prey use in prefur trade populations. In addition, ancient sea otters at the most southerly sites occupied an isotopic niche that was more than twice as large as ancient otters from northerly regions. This likely reflects greater invertebrate prey diversity in southern California relative to northern California. Thus, we suggest the potential dietary niche of sea otters in southern California could be larger than in central and northern California. At two sites, Año Nuevo and Monterey Bay, ancient otters had significantly higher δ15N values than modern populations. Amino acid δ15N data indicated this resulted from shifting baseline isotope values, rather than a change in sea otter trophic ecology. Our results help in better understanding the contemporary ecological role of sea otters and exemplify the strength of combing zooarchaeological and biological information to provide baseline data for conservation efforts.