Homoserine represents a substantial component of pea root exudate that may be important for plant–microbe interactions in the rhizosphere. We identified a gene cluster on plasmid pRL8JI that is required for homoserine utilization by Rhizobium leguminosarum bv. viciae. The genes are arranged as two divergently expressed predicted operons that were induced by L-homoserine, pea root exudate, and were expressed on pea roots. A mutation in gene pRL80083 that prevented utilization of homoserine as a sole carbon and energy source affected the mutant's ability to nodulate peas and lentils competitively. The homoserine gene cluster was present in approximately 47% of natural R. leguminosarum isolates (n = 59) and was strongly correlated with homoserine utilization. Conjugation of pRL8JI to R. leguminosarum 4292 or Agrobacterium tumefaciens UBAPF2 was sufficient for homoserine utilization. The presence of L-homoserine increased conjugation efficiency of pRL8JI from R. leguminosarum to a pRL8JI-cured derivative of R. leguminosarum 1062 and to A. tumefaciens UBAPF2, and induced expression of the plasmid transfer gene trbB; however, there was no difference in conjugation efficiency or trbB expression with A. tumefaciens UBAPF2pRL8-Gm as the donor suggesting that other genes in R. leguminosarum may contribute to regulating conjugation of pRL8 in the presence of homoserine.