Folate is an essential micronutrient that, in mammals, must be obtained from exogenous sources via intestinal absorption. Previous studies have characterized different aspects of the mechanism of the intestinal folate uptake process. Much less, however, is known about regulation of this process. In this study, we examined the effect of dietary folate deficiency on intestinal folate uptake using the rat as an animal model. The results showed that dietary folate deficiency leads to a significant (P < 0.01) and specific upregulation in the transepithelial transport of folic acid. The upregulation in transepithelial folate transport 1) was found to be due to an induction in carrier-mediated folate uptake across the brush-border membrane (BBM) and was mediated via a significant (P < 0.01) increase in the maximal velocity but not the apparent Michaelis constant of the uptake process, 2) was associated with a marked increase in the steady-state mRNA level of reduced folate carrier-1 and in the level of the expressed protein at the intestinal BBM, and 3) was associated with a marked (>10-fold) increase in the activity of the intestinal BBM form of folate hydrolase. Results of this study demonstrate, for the first time, that dietary folate deficiency leads to a marked upregulation in intestinal folate uptake and in the activity of folate hydrolase. Furthermore, the upregulation in folate uptake is associated with an increase in mRNA and protein levels of folate carrier, suggesting possible involvement of a transcriptional regulatory mechanism(s) in the upregulation.