The complete nucleotide sequence of the genome of *Mesorhizobium loti* strain MAFF303099 was determined, consisting of a single chromosome (7,036,071 bp) and two plasmids, pMLa (351,911 bp) and pMLb (208,315 bp). The chromosome contains 6752 potential protein-coding genes, two sets of rRNA genes, and 50 tRNA genes representing 47 tRNA species. Fifty-four percent of the potential protein genes showed sequence similarity to known genes, 21% to hypothetical genes, and 25% had no apparent similarity. A 611-kb DNA segment, identified as a symbiotic island, contains 30 genes for nitrogen fixation and 24 genes for nodulation. The plasmids contain 320 and 209 potential protein-coding genes, respectively, for various biological functions, including ABC-transporter systems, phosphate assimilation, two-component systems, DNA replication, and conjugation. Codon usage analysis suggests that the symbiotic island and plasmids originated and were transmitted from other genetic systems. The study provides insights into the genetic mechanisms underlying symbiotic nitrogen fixation and horizontal gene transfer in *Mesorhizobium loti*.The complete nucleotide sequence of the genome of *Mesorhizobium loti* strain MAFF303099 was determined, consisting of a single chromosome (7,036,071 bp) and two plasmids, pMLa (351,911 bp) and pMLb (208,315 bp). The chromosome contains 6752 potential protein-coding genes, two sets of rRNA genes, and 50 tRNA genes representing 47 tRNA species. Fifty-four percent of the potential protein genes showed sequence similarity to known genes, 21% to hypothetical genes, and 25% had no apparent similarity. A 611-kb DNA segment, identified as a symbiotic island, contains 30 genes for nitrogen fixation and 24 genes for nodulation. The plasmids contain 320 and 209 potential protein-coding genes, respectively, for various biological functions, including ABC-transporter systems, phosphate assimilation, two-component systems, DNA replication, and conjugation. Codon usage analysis suggests that the symbiotic island and plasmids originated and were transmitted from other genetic systems. The study provides insights into the genetic mechanisms underlying symbiotic nitrogen fixation and horizontal gene transfer in *Mesorhizobium loti*.