Abstract Riboflavin (vitamin B2) is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which act as key cofactors of many enzymes, thus has essential roles in cell growth and functions. Animals cannot synthesize riboflavin in situ, the intake, distribution and metabolism of which are mediated by three riboflavin transporters (RFVT1-3). Many mutations in RFVTs cause severe consequences. How RFVTs recognize and transport riboflavin remains largely unknown. Here we describe the cryo-electron microscopy structures of human RFVT2 and RFVT3 in complex with riboflavin in outward-occluded and inward-open states, respectively. Riboflavin is recognized by a conserved binding pocket in the central cavity of RFVTs, whereas two acidic residues in RFVT3 determine its pH-dependent activity. By combining the structural, computational and functional analyses, this study demonstrates the structural basis of riboflavin recognition and provides a structural framework for the mechanistic comprehension of riboflavin recognition, transport, and pathology in human RFVTs.