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Urbanization is one of the most dramatic forms of environmental change with the potential to alter trophic relationships among organisms in cities. In this study, we combine gut-content and stable isotope analyses of brown anole diets with field sampling of arthropods to assess whether and how urbanization-induced variation of basal resources influence trophic niche variation of anole populations inhabiting urban, suburban, and natural habitats. We found clear evidence for trophic niche variation across the urbanization gradient. Stable isotope analyses revealed that urban and suburban anoles had strongly elevated δ13C levels and slightly lower δ15N levels compared to anoles from natural forests— results consistent with increased reliance on grasses across the urbanization gradient. Isotopic niche widths were also greater in urban habitats. However, our analysis of anole diets revealed no difference in the diversity of prey consumed—despite urban habitats having the highest diversity of available prey compared to suburban and natural forest habitats. Overall, our results suggest that much of the observed variation in food web structure results from a shift in the diversity of basal resource pools across the urbanization gradient. The broader effects of a shift towards grass-dominated food webs in urban habitats remains to be understood.