Ensifer (syn. Sinorhizobium) meliloti U143 is an effective nitrogen-fixing strain isolated from Uruguayan soils. For decades, this strain has been used as an inoculant for different alfalfa cultivars. Here we report for the first time a characterization of the U143 elite strain that includes the preliminary genomic sequence, its annotation, and physiological parameters related to its symbiotic efficiency and nitrate respiration capacity. Through Illumina sequencing, the genome of the U143 strain was sequenced. The genome length was 6,801,966 bp, and it contained two megaplasmids, an average GC content of 62.15 %, and 6522 protein-coding sequences. In the symbiotic plasmid, we identified nap, nir, nor, and nos sequences that explain the ability of the U143 strain to respire NO3- in free-living and microaerobic conditions. Field assays performed in two locations for two years showed that alfalfa inoculated with the U143 strain produced 41 % more total shoot dry matter than the non-inoculated control, and between 61.3 % and 66.5 % of shoot N in alfalfa inoculated with strain U143 derived from nitrogen fixation. Display omitted •Ensifer meliloti U143 strain was isolated from Medicago sativa nodules in Uruguayan soils. .•Elite U143 strain has been used as a commercial inoculant for alfalfa cultivars since 1991.•The genome length was 6,807,606 bp, with a GC average content of 62.14 % and 6522 protein-coding sequences.