Many bacteria of the genus Bradyrhizobium are capable of inducing nodules in legumes. In this work, the importance of a type VI secretion system (T6SS) in a symbiotic strain of the genus Bradyrhizobium is described. T6SS of Bradyrhizobium sp. LmicA16 (A16) is necessary for efficient nodulation with Lupinus micranthus and L. angustifolius . A mutant in the gene vgrG, coding for a component of the T6SS nanostructure, induced less nodules and smaller plants than the wild type strain (wt) and was less competitive when co-inoculated with the wt strain. A16 T6SS genes are organized in a 26 kb DNA region in two divergent gene clusters of nine genes each. One of these genes codes for a protein (Tsb1) of unknown function but containing a methyltransferase domain. A tsb1 mutant showed an intermediate symbiotic phenotype regarding vgrG mutant and higher mucoidy and motility than the wt strain in free living conditions. T6SS promoter fusions to the lacZ reporter indicate expression in nodules but not in free living cells grown in different media and conditions. The analysis of nodule structure revealed that the level of nodule colonization was significantly reduced in the mutants with respect to the wt strain.