Riboflavin (RF) is essential for normal cellular functions and health. Humans obtain RF from exogenous sources via intestinal absorption that involves a highly specific carrier-mediated process. We have recently established that the riboflavin transporter-3 (RFVT3) is vital for the normal intestinal RF uptake process, and have characterized certain aspects of its transcriptional regulation. Little is known, however, about how this transporter is regulated at the post-transcriptional level. We address this issue focusing on the role of microRNAs. Using bioinformatics, we identified two potential interacting miRNAs with the human (h) RFVT3- 3'-UTR, and showed (using pmirGLO- hRFVT3-3'-UTR) that the hRFVT3-3'-UTR is indeed a target for miRNA effect. Of the two putative miRNAs identified, miR-423-5p was found to be highly expressed in intestinal epithelial cells, and that its mimic affected luciferase reporter activity of the pmirGLO-hRFVT3-3'-UTR construct, and also lead to inhibition in RF uptake by intestinal epithelial Caco-2 and HuTu-80 cells. Further cells transfected with mutated seed sequences for miR-423-5p showed an abrogation in inhibitory effect of the miR-423-5p mimic on luciferase activity. While miR-423-5p did not affect the level of expression of the hRFVT3 mRNA, it did lead to a significant inhibition in level of expression of its protein. Similarly, miR-423-5p was found to affect the level of expression of the mouse RFVT3 in cultured intestinal enteroids. These findings demonstrate, for the first time, that the RFVT3 is a target for post-transcriptional regulation by miRNAs in intestinal epithelial cells, and that this regulation has functional consequences on intestinal RF uptake.