A considerable amount of the thiamin generated by gut microbiota exists in the form of thiamin pyrophosphate (TPP). We have previously shown that human colonocytes possess an efficient carrier-mediated uptake process for TPP that involves the SLC44A4 system and this uptake process is adaptively-regulated by prevailing extracellular TPP level. Little is known about the molecular mechanisms that mediate this adaptive regulation. We addressed this issue using human-derived colonic epithelial NCM460 cells and mouse colonoids as models. Maintaining NCM460 cells in the presence of high level of TPP (1 mM) for short (2 days) and long (9 days) periods was found to lead to a significant reduction in ³H-TPP uptake compared to cells maintained in its absence. Short-term exposure showed no changes in level of expression of SLC44A4 protein in total cell homogenate (although there was a decreased expression in the membrane fraction), mRNA and promoter activity. However, a significant reduction in the level of expression of the SLC44A4 protein, mRNA and promoter activity was observed upon long-term maintenance with the substrate. Similar changes in Slc44a4 mRNA expression were observed when mouse colonoids were maintained with TPP for short- and long-terms. Expression of the transcription factors ELF3 and CREB-1, (which drive the SLC44A4 promoter), following long-term exposure was unchanged; but their binding affinity to the promoter was decreased and specific histone modifications were also observed. These studies demonstrate that, depending on the period of exposure, different mechanisms are involved in the adaptive regulation of colonic TPP uptake by extracellular substrate level.