Provided is a gene targeting vector that enables highly efficient gene targeting. The gene targeting vector has a structure comprising a positive selection marker flanked by a DNA homologous to a 5′-upstream region of a target site and a DNA homologous to a 3′-downstream region of the target site, wherein a splice acceptor site and a DNA sequence allowing for bicistronic expression are added 5′-upstream of the positive selection marker, and another splice acceptor site is also added 5′-upstream of the DNA homologous to the 5′-upstream region of the target site.