We investigated whether cephalexin transport in Caco-2 cells is regulated by insulin. After the insulin pretreatment, cephalexin uptake, and transport as well as PEPT1 mRNA and protein expression in the cells were measured. Cephalexin uptake was significantly increased by the insulin pretreatment. Insulin significantly increased cephalexin saturable uptake, but had no significant effect on the non-saturable one. PEPT1 protein expression on the apical membrane, but not PEPT1 mRNA expression, was increased by the insulin pretreatment. The enhancement of cephalexin uptake by the insulin pretreatment was inhibited by genistein, a tyrosine kinase inhibitor, and colchicine, an agent that disrupts protein translocation. Apical-to-basolateral transport of cephalexin has increased by the insulin pretreatment at the apical side and long-term insulin pretreatment at the basolateral side. It is considered that insulin mainly binds to its receptor on the apical and basolateral membranes, thereby promoting PEPT1 translocation from the intracellular pool to the apical membrane surface; consequently, PEPT1 protein expression on the apical membrane is increased.