The conjugative plasmid, pLS20, isolated from Bacillus subtilis natto, has an outstanding capacity for rapid self-transfer. In addition, it can function as a helper plasmid, mediating the mobilization of an independently replicating co-resident plasmid. In this study, the oriT sequence of pLS20cat (oriT ) was eliminated to obtain the plasmid, pLS20catΔoriT. This resulted in the complete loss of the conjugative transfer of the plasmid but still allowed it to mobilize a co-resident mobilizable plasmid. Moreover, pLS20catΔoriT was able to mobilize longer DNA segments, up to 113 kb of chromosomal DNA containing oriT , after mixing the liquid cultures of the donor and recipient for only 15 min. The chromosomal DNA mobilization mediated by pLS20catΔoriT will allow us to develop a novel genetic tool for the rapid, easy, and repetitive mobilization of longer DNA segments into a recipient chromosome.