Six conserved, charged amino acids within membrane spans in rice sucrose transporter OsSUT1 were identified using a three-dimensional structural model based on the crystal structures of three major facilitator superfamily (MFS) proteins: LacY, GlpT, and EmrD. These positions in OsSUT1 were selected for mutagenesis and biochemical assays. Among the six mutants, D177N completely lost transport function, D331N retained only a small fraction of sucrose uptake activity (2.3% of that of the wild type), and R335H and E336Q also displayed a substantial decrease in transport activity. D329N functioned as well as wild-type OsSUT1. R188K did not transport sucrose but showed a H+ leak that was inhibited by sucrose, indicating that R188K had uncoupled sucrose and H+ translocation. This demonstrates that charged amino acids within membrane spans are important for the transport mechanism of OsSUT1 as they are in lactose permease.