Drug delivery systems (DDSs) incorporating bacterial minicells have been evaluated as a very powerful tool in view of biocompatibility. However, limited studies have been carried out on these systems, mainly using minicells from Salmonella sp. and Escherichia coli. Thus, we generated a new minicell-producing strain from an endotoxin-free Corynebacterium glutamicum by the inactivation of genes related to cell division. The two knockout strains, ΔparA and Δncgl1366, showed distinct abilities to produce minicells. The resulting minicells were purified via sequential antibiotic treatments and centrifugations, which resulted in reproducible yields.