Type III secretion systems (T3SS) have been found in several species of rhizobia. Proteins (termed effectors) secreted by this system are involved in host-range determination and influence nodulation efficiency. Mesorhizobium loti MAFF303099 possesses a functional T3SS in its symbiotic island whose expression is induced by flavonoids. As in other rhizobia, conserved cis-elements (tts box) were found in the promoter regions of genes or operons encoding T3SS components. Using a bioinformatics approach, we searched for other tts-box-controlled genes, and confirmed this transcriptional regulation for some of them using lacZ fusions to the predicted promoter regions. Translational fusions to a reporter peptide were created to demonstrate T3SS-mediated secretion of two new MAFF303099 effectors. Finally, we showed that mutation of the M. loti MAFF303099 T3SS affects its competitiveness on Lotus glaber and investigated, at the molecular level, responses of the model legume L. japonicus to the T3SS.