What an animal eats and how it digests its food determines that animal’s contribution to its ecosystem. However, the nutritional physiology (including digestion) of animals is commonly overlooked, even though such data are needed for conservation of many ecosystems. This is particularly true for wild fishes in comparison to terrestrial vertebrates or even fishes farmed for aquaculture. The goal of my dissertation is to understand how prickleback fishes specialize to use specific resources, and how their nutritional physiology impacts their roles in their environment. I aimed to address the research gap in our understanding of dietary specialization and fish nutritional physiology, which are topics that I briefly review in the first chapter of my dissertation.In the second chapter, I integrated nutritional physiology and transcriptomics to further our understanding of digestive system plasticity in response to dietary perturbations. This study showed that prickleback fishes with different diets can respond to dietary perturbations in different ways. Our dataset elegantly shows how gut length of fishes change with dietary perturbations, even within a single species over a four-week feeding experiment. Although there were hundreds of genes differentially expressed among the three diet groups in the intestine (pyloric ceca and mid-intestine), three of the four species didn’t appreciably alter gene expression in the liver in response to different diets in the laboratory. Thus, we find a species-specific pattern in liver gene expression and metabolic pathways within prickleback fishes. In the third chapter of my dissertation, I aimed to advance our understanding of the factors that can influence gut physiology and function to provide insight into ecological adaptations and potential impacts on ecosystem dynamics. I investigated the effects of different diets that vary in protein content, on the physiology, host gene expression, and gut microbiome of Cebidichthys violaceus, a marine herbivorous fish. This integrative study highlighted the complex interactions between diet, gut physiology, gene expression and the hindgut microbiome in a marine herbivorous fish. Interactions between gut microbes and animal hosts impact the physiology and health of animals, yet we lack a thorough understanding of the determinants of gut microbial community structure. In chapter four of my dissertation, I examined the factors that shape the gut microbial communities of prickleback fishes within the context of their environment, diet, and genetics. We discovered that changes in the gut microbiome were linked more to individual differences and species identity within closely-related fishes living in the same area, rather than being primarily influenced by diet or location. In summary, dietary perturbations can alter the gut physiology, transcriptome, and microbiome of prickleback fishes. My dissertation work advances our understanding of dietary specialization in vertebrates and fish nutritional physiology.