Cre/lox system derived from P1 bacteriaphage can quickly and effectively achieve gene insertion, deletion, replacement, and inversion by means of site-specific recombination. As one of the most important tools for gene targeting at present, Cre/lox system has been widely used in Arabidopsis thaliana, Oryza sativa L., Mus musculus, Drosophila melanogaster, Danio rerio, and other higher eukaryotic organisms. This review roundly described the basic profile of Cre/lox system, and its application in higher eukaryotes. In addition, we also discussed the main problems and developmental trend of the Cre/lox system in this review, which can be a good reference for using Cre/lox system to realize the gene manipulations of the different high eukaryotic organisms.