Recent studies on the intestinal uptake of the organic cation 1-methyl-4-phenylpyridinium (MPP+) showed that transport of this compound occurs through human extraneuronal monoamine transporter (hEMT). Moreover, it was recently described that alkaline phosphatase (ALP), an ecto-phosphatase anchored to the plasma membrane and able to dephosphorylate extracellular substrates or cell-surface proteins, is directly or indirectly involved in the modulation of MPP+ uptake by Caco-2 cells. The present study investigated a putative modulation of MPP+ intestinal apical uptake and ecto-ALP activity by thiamine (T+) and thiamine pyrophosphate (TPP, a T+ dietary precursor). For this purpose, we used Caco-2 cells, an enterocyte-like cell line derived from a human colonic adenocarcinoma, as an intestinal model. Ecto-ALP activity and N-[methyl-3H]-4-phenylpyridinium acetate (3H-MPP+) uptake were evaluated in intact Caco-2 cells. T+ and TPP were able to increase ecto-ALP activity, with an equal potency, and to decrease 3H-MPP+ apical uptake, with a similar potency. The effects of both compounds on ecto-ALP activity and 3H-MPP+ uptake were concentration-dependent. The results suggest that the effect of T+ and TPP on ecto-ALP activity may lead to inhibition of the intestinal absorption of other organic cations present in the diet. Another important conclusion is that the intestinal absorption of T+ may occur through hEMT, in Caco-2 cells.