We have previously demonstrated that leptin inhibits the sugars absorption in rat intestine in vitro and in vivo acting from the lumen, with the involvement of PKA and mainly PKC. We also found that leptin receptors are expressed in both the apical and basolateral membrane of enterocytes. In the present work we have studied the effect of leptin on the absorption of α-methyl-glucoside (MG), glutamine and β-alanine by the human intestinal epithelial cell line Caco-2 and the possible implication of PKC and PKA. The Caco-2 monolayer was formed on plates (access from the apical membrane) and filters (access from the basolateral side). The apical uptake of the substrate was measured in the absence or presence of leptin (0.2 and 8 nM) after 5, 15 and 30 min incubation. The involvement of PKA or PKC was studied by preincubating the cells for 30 min with the respective inhibitors H-89 (1 μM) or chelerythrine (chel) (2 μM). Leptin present in the apical side inhibited by 25 % the uptake of 0.1 mM MG (substrate of SGLT1) after 5 and 30 min. Interestingly, after 15 min, 0.2 nM leptin did not modify sugar uptake whereas 8 nM increased it by 20 %. Both H-89 and chel reversed this increase, indicating the implication of PKA and PKC on leptin effect. At 5 and 30 min, however, the inhibitors seemed to potentiate the decreasing effect of the hormone. On the other hand, leptin present in the basal compartment inhibited, by 30 %, MG uptake only at 8 nM after 30 min. Absorption of 0.1 mM β-Ala was measured at pH 6 in the presence of Na⁺, which is the optimal condition for the activity of its transporter PAT1. The effect of apical leptin on β-Ala uptake followed the same pattern as for MG at the three incubation times. However, leptin acting from the basolateral side did not modify the amino acid uptake. Leptin inhibition of β-Ala uptake at 30 min was completely reversed by H-89, indicating the involvement of PKA, whereas chel did not show any effect. Finally, uptake of 0.1 mM Gln (substrate of ASCT2 and B⁰AT1) was also inhibited 40% by apical leptin at 5, 15 and 30 min and by basolateral leptin after 15 min (15%). As it happened for MG, both H-89 and chel increased leptin inhibitory effect at 30 min, indicating that neither PKA nor PKC are implicated in leptin action at this incubation time. In summary, leptin regulates the activity of sugar and amino acid transporters in Caco-2 cells, acting mainly from the apical membrane, with implication of PKA and PKC.