Protein is the most costly nutrient in fish feed, and while diets offered in the early stages of development typically have high levels of CP, they do not always correspond to the real requirements of the animals. Thus, research that seeks to learn the true nutritional requirements of fish is fundamental to improving commercial fish culture. The present study evaluated the protein requirements of Nile tilapia (Oreochromis niloticus) under larviculture. Fish performance, gene expression for digestive enzymes and their enzymatic activity and stress response to air exposure were analyzed. Four experimental diets differing in CP level were formulated: 30%, 36%, 42% and 48%. Fish larvae were fed the experimental diets during development and sampled 10, 20 and 30 days after the beginning of the experiment for performance, gene expression and enzymatic activity. At sampling time 30, stress resistance was also evaluated by means of an air exposure test. At sampling time 10, CP levels between 36% and 48% could be used for a better performance. During this period, pepsinogen expression was greater for 30% CP, intermediate for 42% and lower for 36% and 48%. After this initial period, diets of between 30% and 42% CP are recommended for better performance. At sampling time 20, gene expression for digestive enzymes and their enzymatic activity were similar for all diets tested. At sampling time 30, the diet of 42% CP induced both greater pepsinogen expression and pepsin activity. Survival after the air exposure test after 30 days of feeding was influenced by CP level in the diet, with the highest survival being for fish fed with 36% CP. Taken together, the present results demonstrate that dietary CP influences digestive enzyme gene expression and activity, and suggest that the best CP levels for Nile tilapia larviculture vary depending on larval stage.