The regulation of glycerol permeability in the gastrointestinal tract is crucial to control fat deposition, lipolysis and gluconeogenesis. Knowing that the amino acid glutamine is a physiological regulator of gluconeogenesis, whereas cystine promotes adiposity, herein we investigated the effects of dietary supplementation with glutamine and cystine on the serum biochemical parameters of piglets fed on amino acid-enriched diets, as well as on the transcriptional profile of membrane water and glycerol channels aquaporins (AQPs) in the ileum portion of the small intestine and its impact on intestinal permeability. Twenty male piglets with an initial body weight of 8.8 ± 0.89 kg were allocated to four dietary treatments (n = 5) and received, during a four week-period, a basal diet without supplementation (control) or supplemented with 8 kg/ton of glutamine (Gln), cystine (Cys) or the combination of the two amino acids in equal proportions (Gln + Cys). Most biochemical parameters were found improved in piglets fed Gln and Cys diet. mRNA levels of AQP3 were found predominant over the others. Both amino acids, individually or combined, were responsible for a consistent downregulation of AQP1, AQP7 and AQP10, without impacting on water permeability. Conversely, Cys enriched diet upregulated AQP3 enhancing basolateral membranes glycerol permeability and downregulating glycerol kinase (GK) of intestinal cells. Altogether, our data reveal that amino acids dietary supplementation can modulate intestinal AQPs expression and unveil AQP3 as a promising target for adipogenesis regulation.