Background <p>Understanding what shapes variation in organisms’ capacity to utilize novel resources is essential to predicting how species will respond to environmental change. For herbivores, exposure to toxic phytochemicals in novel plants may limit persistence in new habitats. We investigated the behavioral, physiological, genetic, and microbial consequences of diet switching in two closely related species of rodent herbivores that each consume differentially toxic plants in their native habitat, and that maintain different dietary strategies (i.e., relative dietary specialist versus relative generalist).</p> Results <p>In reciprocal laboratory feeding trials, we exposed wild-caught woodrats (genus <i>Neotoma</i>) to toxins characteristic of either familiar or novel plant secondary compounds. We measured changes in food and water intake, locomotor activity, gut microbial composition, and gene expression across the digestive tract following feeding trials. The dietary generalist responded minimally, but the specialist responded strongly when exposed to the novel diet. This response included behavioral and genetic components including increased water intake, reduction in locomotor activity, increased differential expression of detoxification genes, and a greater shift in gut microbial composition.</p> Conclusions <p>The dietary specialist exhibited a strong response to diet switching that corresponded with ecologically relevant shifts in behavior and physiology that would have negative fitness consequences. Although the dietary specialist had a strong genetic and microbial response to novel plant secondary compounds, this response would likely be insufficient to overcome the immediate challenge of exposure to novel dietary toxins in the wild. Our results underscore the link between feeding strategy and the capacity to shift to novel dietary resources in response to environmental change.</p>