This study uses stable isotope analyses (d 13 C and d 15 N) of human bone collagen to reconstruct the diet of three Romano-British (first to early fifth century AD) populations from Gloucestershire in South West England. Gloucestershire was an important part of Roman Britain with two major administrative centres at Gloucester (Glevum) and Cirencester (Corinium) and numerous smaller settlements and farmsteads. To investigate potential dietary differences between the rural and urban populations of Roman Gloucestershire, we compared human bone collagen stable isotope values from 32 individuals from urban Gloucester with those of 46 individuals from two rural cemeteries at Horcott Quarry and Cotswold Community, respectively. Seven individuals from urban Gloucester were buried in a mass grave; all others were buried in single inhumations. Results show small but significant differences in stable isotope ratios between the urban and rural populations which indicate that the urban population might have consumed slightly more marine and/or freshwater resources than the people living in the rural communities. We interpret this difference as a direct reflection of Rome’s influence on Gloucester’s population and the town’s economic status. Subtle differences in stable isotope ratios were also observed at the site level, as burial practice does correlate with diet in some cases. Overall, the results from this study demonstrate that diet, as reconstructed through stable isotope analysis, is a very sensitive, if settlement-specific, indicator of social differentiation and culture change.