Robust gene disruption and silencing methods are required for metabolic engineering. Gene disruption methods are suitable for the complete absence of gene function, but it is problematic when target genes are crucial for cell growth. On the other hand, gene silencing is applicable even when target genes are essential for growth. We here describe a conditional gene silencing method where up to four genes are silenced simultaneously using antisense RNAs in Escherichia coli. To express multiple antisense RNAs in parallel, we constructed four IPTG-inducible vectors carrying compatible replication origins and different selection markers. As antisense RNAs, a paired-termini design was employed, which carries flanking inverted repeats to create paired double stranded RNA termini. Such an RNA design was much more effective compared to conventional ones. Also, it has been shown that using the method, altering metabolic pathways to fit industrial purposes is easily achieved. The multiple gene silencing method is useful for investigating gene function and gene interaction and for metabolic engineering.