On-chip transformation of Escherichia coli cells was accomplished for the first time using a microbial array chip. The continuous E. coli transformation procedures were performed on a chip in which the microcompartment was composed of PDMS microfluidic channels and a silicon substrate predeposited with different plasmid DNAs. The PDMS microfluidic device enabled the parallel transformation of E. coli cells with various plasmid DNAs by separating each transformation area. The phenotypic differences reflecting different plasmid DNAs were identified by various approaches such as colorimetry, fluorometry, and electrochemical methods. This microbial array chip could become a versatile tool for many cell biological applications.