Minicells have been studied as drug delivery with highly significant anti-tumor effects in vivo. Minicells were produced by mutant bacteria without min locus. In this study, the minicell generation was done in the glucose condition in combination with different antibiotic concentrations when culturing the Escherichia coli BL21(DE3)plysS harboring pET 21(a+) and pET 28(a+) inserted with minD gene of Lactobacillus acidophillus VTCC-B-871. The minD gene gives a filamentous phenotype in 1% glucose and ampicillin at concentration at 100mg/ml for the host cells harboring pET 21(a+)/minD and kanamycin at 50mg/ml for the host cells harboring pET 28(a+)/minD. However, the filaments changed into minicells when chloramphenicol presented in the culture with the long incubation time to 24 hours. Especially, glucose could induce the expression of MinD. The morphology differentiation was observed by microscope. The study supported the minicell generation with high rate and enough amount for drug delivery.