ABSTRACT: Plant nutritional properties, physical defenses, and chemical defenses impact herbivore feeding decisions. While herbivores often prefer plants enriched with nitrogen, less is known about how phosphorus impacts plant-herbivore interactions. In this study, we investigated how seagrass-herbivore interactions vary along a natural gradient in phosphorus availability, using Thalassia testudinum (turtle grass) collected from three sites along the gradient (Weeki Wachee = low phosphorus and nitrogen, Homosassa = high phosphorus and nitrogen, Crystal River = high phosphorus and low nitrogen) and two common herbivores, Lytechinus variegatus (variegated sea urchin) and Nicholsina usta (emerald parrotfish). Thalassia testudinum plants from the phosphorus-poor site (Weeki Wachee) exhibited increased physical and chemical anti-herbivore defenses. Plants in Weeki Wachee had higher leaf fiber content, lower specific leaf area, and up to 2x higher concentrations of phenolic acids and condensed tannins. Both L. variegatus and N. usta avoided consuming the plants from Weeki Wachee, preferring plants from sites with higher nutrient availability. However, feeding preferences varied between the two herbivore species, with L. variegatus often consuming similar amounts of the plants from the two phosphorus-rich sites (Homosassa and Crystal River) and N. usta preferring the plants from Homosassa, which had the highest concentrations of both phosphorus and nitrogen. These findings suggest that plants at the phosphorus-poor site invest more resources in anti-herbivore defenses, and this higher investment in defenses, along with lower leaf tissue phosphorus concentrations, deterred multiple herbivore species.