1-Deoxymannojirimycin (dMM), a specific alpha-mannosidase I inhibitor, completely blocks the conversion of Man9-8GlcNAc2 into Man7-5-GlcNAc2 in both differentiated and undifferentiated human adenocarcinoma HT-29 cells. Besides this well known effect on N-glycan trimming, we describe here a novel effect of this inhibitor on the D-[2-3H]mannose uptake that is exclusively observed in differentiated intestinal cells, i.e. cells that display a functional apical brush border membrane. This inhibition of D-[2-3H]mannose uptake was shown to be dose-dependent and reversible. Moreover, using microsomal fractions we showed that this effect depends only on the integrity of the brush border and is unrelated to the classical inhibitory effect of dMM on N-glycan processing. Furthermore, another N-glycan trimming inhibitor 1-deoxynojirimycin, an epimer of dMM, did not interfere with D-[2-3H]mannose uptake. This observation was in good agreement with the specificity of the effect induced by dMM. These results demonstrate a novel effect of dMM on highly differentiated intestinal cells and suggest that a carrier-mediated mannose transport could exist in those cells. Such an interaction between cell morphology and the biological effect of dMM should lead to a careful use of drugs acting on N-glycan processing.