The foodborne bacteriumStaphylococcus equorumstrain KS1030 harbours plasmid pSELNU1, which encodes a lincomycin resistance gene. pSELNU1 undergoes horizontal transfer between bacterial strains, thus spreading antibiotic resistance. However, the genes required for horizontal plasmid transfer are not encoded in pSELNU1. Interestingly, a relaxase gene, a type of gene related to horizontal plasmid transfer, is encoded in another plasmid ofS. equorumKS1030, pKS1030-3. The complete genome of pKS1030-3 is 13,583 bp long and encodes genes for plasmid replication, biofilm formation (theicaoperon), and horizontal gene transfer. The replication system of pKS1030-3 possesses the replication protein-encoding generepB, a double-stranded origin of replication, and two single-stranded origins of replication. Theicaoperon, relaxase gene, and a mobilization protein-encoding gene were detected in pKS1030-3 strain-specifically. When expressed inS. aureusRN4220, theicaoperon and relaxase operon of pKS1030-3 conferred biofilm formation ability and horizontal gene transfer ability, respectively. The results of our analyses show that the horizontal transfer of pSELNU1 ofS. equorumstrain KS1030 depends on the relaxase encoded by pKS1030-3, which is therefore trans-acting. Genes encoded in pKS1030-3 contribute to important strain-specific properties ofS. equorumKS1030. These results could contribute to preventing the horizontal transfer of antibiotic resistance genes in food.