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Simple Summary: The ammonia content in aquaculture waterbodies can easily achieve a relatively high level, largely due to the excretion of intensively farmed aquatic animals and the decomposition of residual food particles. Excess ammonia may exert prominent toxicities to all aquatic species. Many freshwater turtles have become economically important aquaculture species in some countries. The evaluation of the toxic effects of ammonia on these cultured turtle species is rarely considered, despite having been extensively conducted in many invertebrates and fish. Various omics techniques can be applied to comprehensively analyze diverse biological responses at different levels in pollutant-exposed individuals. In order to increase our understanding of the physiological changes under ammonia stress in cultured turtle species, we exposed juveniles of the red-eared slider turtle (Trachemys scripta elegans) to different concentrations (0, 0.3, 3.0, and 20.0 mg/L) of ammonium chloride solution for 30 days, and then measured their swimming performance, growth rate, gut microbial composition, and liver metabolic profiling. No significant change in swimming ability was found in ammonia-exposed turtles, but a relatively low growth rate was exhibited in the 20.0 mg/L ammonia-exposed turtles. Although no significant differences in the alpha and beta diversity measures of the gut microbial community were observed, the microbial composition seemed to be different among different treatment groups. Some pathogenic bacteria were found to increase in ammonia-exposed turtles. Liver metabolic profiling based on liquid chromatography–mass spectrometry showed that a series of metabolites (such as leucine, valine, arginine, glutamine, adenosine diphosphate, glyceric acid, myo-inositol, gamma-aminobutyric acid, etc.) were significantly altered in ammonia-exposed turtles. Accordingly, ammonia exposure might alter the health status of guts due to increased pathogenic bacteria, and disturb multiple metabolic pathways (such as amino acid, nucleotide, lipid metabolism, energy metabolism, etc.) in juveniles of T. scripta elegans. As the most common pollutant in aquaculture systems, the toxic effects of ammonia have been extensively explored in cultured fish, molluscs, and crustaceans, but have rarely been considered in turtle species. In this study, juveniles of the invasive turtle, Trachemys scripta elegans, were exposed to different ammonia levels (0, 0.3, 3.0, and 20.0 mg/L) for 30 days to evaluate the physiological, gut microbiomic, and liver metabolomic responses to ammonia in this turtle species. Except for a relatively low growth rate of turtles exposed to the highest concentration, ammonia exposure had no significant impact on the locomotor ability and gut microbial diversity of turtles. However, the composition of the microbial community could be altered, with some pathogenic bacteria being increased in ammonia-exposed turtles, which might indicate the change in their health status. Furthermore, hepatic metabolite profiles via liquid chromatography–mass spectrometry revealed extensive metabolic perturbations, despite being primarily involved in amino acid biosynthesis and metabolism. Overall, our results show that ammonia exposure causes gut dysbacteriosis and disturbs various metabolic pathways in aquatic turtle species. Considering discrepant defense mechanisms, the toxic impacts of ammonia at environmentally relevant concentrations on physiological performance might be less pronounced in turtles compared with fish and other invertebrates. [ABSTRACT FROM AUTHOR]