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The Neotropical catfish Pseudoplatystoma punctifer is a promising candidate species for aquaculture diversification in the Amazon basin. However, optimized feeding strategies are needed to improve growth and survival during the early life stages. In order to determine the nutritional needs of early juveniles, the influence of four diets containing different protein and lipid content in their digestive physiology and performance was evaluated. To do so, the gene expression and the activity of the main digestive enzymes, the histology of the digestive organs, and the incorporation of the dietary macronutrients in the body tissue of fish fed the different diets were analyzed. The diet containing the highest protein and lipid content promoted the fastest development of the digestive capacities, resulting in a better growth and survival that increased sixfold and twofold, respectively, in comparison to previous feeding protocols. Lipids were favored over carbohydrates as a source of energy and excessive carbohydrate content led to unbalanced lipid metabolism and fat deposition in the liver. This study provided new knowledge on the molecular and biochemical mechanisms underlying nutrient digestion in this species and on the nutritional needs of early juveniles, which represents a significant contribution towards the establishment of its commercial culture.