The putative hexose transporter gene fst1 in Fusarium verticillioides was identified previously by microarray analysis as a gene that was more highly expressed during colonization of autoclaved maize endosperm than germ. In contrast to a previous study, in which disruption of fst1 did not affect growth of the pathogen on autoclaved maize kernels, in the current study, we demonstrated that disruption of fst1 delayed growth and symptom development on wounded maize ears. Characterization of the fst1 promoter revealed that regulation of fst1 expression was similar to that of fumonisin biosynthetic (fum) genes; expression was highest during growth on endosperm tissue and repressed by elevated concentrations of ammonium in the growth medium. With a fluorescent tag attached to FST1, the protein localized transiently to the periphery of the cells near the plasma membrane and in vacuole-like structures, suggesting that membrane-localized FST1 was internalized and degraded in vacuoles. Expression of fst1 in a yeast strain lacking hexose transporter genes did not complement the yeast mutation, suggesting that FST1 does not transport glucose, fructose, or mannose. The results indicate a functional role for FST1 in pathogenesis during the colonization of living kernels.