Flavin mononucleotide (
FMN) and
flavin adenine dinucleotide (
FAD) are essential
riboflavin-derived cofactors involved in a myriad of redox reactions across all forms of life. Nevertheless, the basis of
flavin acquisition strategies by
riboflavin auxotrophic pathogens remains poorly defined. In this study, we examined how the facultative intracellular pathogen Listeria monocytogenes, a
riboflavin auxotroph, acquires
flavins during
infection. A L. monocytogenes mutant lacking the putative
riboflavin transporter (RibU) was completely avirulent in mice but had no detectable growth defect in nutrient-rich media. However, unlike wild type, the RibU mutant was unable to grow in defined media supplemented with
FMN or
FAD or to replicate in macrophages starved for
riboflavin. Consistent with RibU functioning to scavenge
FMN and
FAD inside host cells, a mutant unable to convert
riboflavin to
FMN or
FAD retained virulence and grew in cultured macrophages and in spleens and livers of infected mice. However, this
FMN- and
FAD-requiring strain was unable to grow in the gallbladder or intestines, where L. monocytogenes normally grows extracellularly, suggesting that these sites do not contain sufficient
flavin cofactors to promote replication. Thus, by deleting genes required to synthesize
FMN and
FAD, we converted L. monocytogenes from a facultative to an obligate intracellular pathogen. Collectively, these data indicate that L. monocytogenes requires
riboflavin to grow extracellularly in vivo but scavenges
FMN and
FAD to grow in host cells.