In drug absorption and permeability experiments, an unstirred water layer (UWL) is known to cause differences in the estimated permeability of drugs between in vitro and in vivo experiments. Therefore, it is necessary to develop a new method to allow for accurate measurements of in vitro drug absorption through the reduction of the UWL effect. Previously, we have developed an artificial intestinal tract that mimics the tubular structure of the human intestine and enables study of drug absorption under flow conditions. In order to determine whether our artificial intestinal tract has the potential to reduce the effect of a UWL on drug absorption, the present study evaluated drug absorption in Caco-2 cells using this artificial system. The viability and tight junction structure of Caco-2 cells on the artificial intestinal tract were intact during perfusion. The cumulative amount of the highly lipophilic drugs imipramine and chlorpromazine accumulated in Caco-2 cells cultured on the cell culture plate was 1.5 times higher under mechanical agitation, whereas that of cells on the artificial intestinal tract was 6.5 times higher when internal flow was applied. In addition, the cumulative amounts of 5-aminosalicylic acid and clonidine, drugs with low lipophilicity, accumulated in Caco-2 cells on the artificial intestinal tract were unchanged by internal flow. These results indicate that the artificial intestinal tract enables effective reduction of the UWL thickness at the Caco-2 cell-surface, and allows evaluation of in vitro drug absorption under conditions similar to those found in vivo .