Hypoxia exerts profound effects on cell physiology, but its effect on colonic uptake of the microbiota-generated forms of vitamin B1 (i.e., thiamin pyrophosphate [TPP] and free thiamine) has not been described. Here, we used human colonic epithelial NCM460 cells and human differentiated colonoid monolayers as in vitro and ex vivo models, respectively, and were subjected to either chamber (1% O2, 5% CO2, and 94% N2) or chemically (desferrioxamine; 250 μM)-induced hypoxia followed by determination of different physiological-molecular parameters. We showed that hypoxia causes significant inhibition in TPP and free thiamin uptake by colonic NCM460 cells and colonoid monolayers; it also caused a significant reduction in the expression of TPP (SLC44A4) and free thiamin (SLC19A2 and SLC19A3) transporters and in activity of their gene promoters. Furthermore, hypoxia caused a significant induction in levels of hypoxia-inducible transcription factor (HIF)-1α but not HIF-2α. Knocking down HIF-1α using gene-specific siRNAs in NCM460 cells maintained under hypoxic conditions, on the other hand, led to a significant reversal in the inhibitory effect of hypoxia on TPP and free thiamin uptake as well as on the expression of their transporters. Finally, a marked reduction in level of expression of the nuclear factors cAMP responsive element-binding protein 1 and gut-enriched Krüppel-like factor 4 (required for activity of SLC44A4 and SLC19A2 promoters, respectively) was observed under hypoxic conditions. In summary, hypoxia causes severe inhibition in colonic TPP and free thiamin uptake that is mediated at least in part via HIF-1α-mediated transcriptional mechanisms affecting their respective transporters.