Arginine (Arg), lysine (Lys), and methionine (Met) can be used to support the health status of turkeys. The present study investigated selected performance, gut integrity, and immunological parameters in turkeys reared in optimal or challenge conditions. The experiment lasted for 28 days, and it had a completely randomized 2 × 3 factorial design with two levels of dietary Arg, Lys and Met (high or low) and challenge with Clostridium perfringens (C. perfringens), Escherichia coli lipopolysaccharide (LPS) or no challenge (placebo). Increased dietary levels of Arg, Lys and Met had a beneficial effect on turkey performance and immunological parameters, and it improved selected indicators responsible for maintaining gut integrity in different challenge conditions. Under optimal conditions (with no challenge), high ArgLysMet diets did not compromise bird performance and they improved selected performance parameters in challenged birds. The immune system of turkeys was not excessively stimulated by high ArgLysMet diets, which did not disrupt the redox balance and had no negative effect on gut integrity. High ArgLysMet diets increased the expression levels of selected genes encoding nutrient transporters and tight junction proteins. However, the influence exerted by different dietary inclusion levels of Arg, Lys and Met on gut integrity was largely determined by the stressor (C. perfringens vs. LPS). Further studies are required to investigate the role of Arg, Lys and Met levels in the diet on the immune response, gut function and performance of turkeys in different challenge conditions.