Gene targeting via homologous recombination is a powerful tool for studying gene function, but the targeting efficiency in human cell lines is too low for generating knockout mutants. Several cell lines null for the gene responsible for Bloom syndrome, BLM, have shown elevated targeting efficiencies. Therefore, we reasoned that gene targeting would be enhanced by transient suppression of BLM expression by RNA interference. To test this, we constructed a gene correction assay system to measure gene targeting frequencies using a disrupted hypoxanthine phosphoribosyltransferase (HPRT) locus in the human HT1080 cell line, and examined the effect of small interfering RNA (siRNA) for BLM on gene targeting. When HPRT-null cells pretreated with BLM siRNA were co-transfected with the siRNA and a gene correction vector, the gene targeting frequency was elevated three-fold, while the random integration frequency was marginally affected. Remarkably, in BLM heterozygous (+/-) cells derived from HPRT-null cells, the BLM siRNA treatment gave more than five-fold higher targeting frequencies, even with one-tenth the amount of BLM siRNA used for BLM+/+ cells. Furthermore, in the human pre-B cell line Nalm-6, the siRNA treatment enhanced gene targeting 6.3-fold and > 5.8-fold at the HPRT and adenine phosphoribosyltransferase (APRT) loci, respectively. These results indicate that transient suppression of BLM expression by siRNA stimulates gene targeting in human cells, facilitating a further improvement of gene targeting protocols for human cell lines.