This study aimed to investigate the gastrointestinal stability and the cellular uptake characteristics of l-valyl-ara-C, a peptidomimetic prodrug of ara-C (cytarabine). After the synthesis of l-valyl-ara-C via the incorporation of l-valine into the N4-amino group of the cytosine ring in ara-C, the gastrointestinal stability of l-valyl-ara-C was examined using artificial gastric juice and artificial intestinal fluids. The cellular uptake characteristics of l-valyl-ara-C were also examined in Caco-2 cells. The disappearance half-life of l-valyl-ara-C was 2.2 h in artificial gastric juice, while the degradation of l-valyl-ara-C was negligible in artificial intestinal fluid and also in the supernatant above the Caco-2 cell monolayer during the 2-h incubation. The cellular accumulation of l-valyl-ara-C was 5-fold higher than that of ara-C in Caco-2 cells. Furthermore, the cellular uptake of l-valyl-ara-C did not increase proportionally to the increase in drug concentration. The cellular accumulation of l-valyl-ara-C was significantly reduced in the presence of uridine, p-aminohippurate, tetraethylammonium and small dipeptides, while it was not changed in the presence of l-valine and benzoic acid, suggesting that l-valyl-ara-C could interact with multiple uptake transporters, including peptide transporters, organic anion and cation transporters and nucleoside transporters, but might not interact with amino acid transporters. In conclusion, l-valyl-ara-C could be effective to improve the oral absorption of ara-C via the carrier-mediated transport pathway.