PCR-based mutagenesis is a cornerstone of molecular biology and protein engineering studies. Here we describe a rapid and highly efficient mutagenesis method using type IIs restriction enzymes. A template gene is amplified into two separate PCR fragments using two pairs of anchor and mutagenic primers. Mutated sequences are located near the recognition site of a type IIs restriction enzyme. After digestion of two fragments with a type IIs enzyme, exposed cohesive ends, which are complementary to each other, are then ligated together to generate a mutated gene. We applied this method to introduce multiple site-directed mutations in EGFP and Bcl-2 family genes, and observed perfect mutagenesis efficiency at the desired sites. This efficient and cost-effective mutagenesis method can be applied to a wide variety of structure-function studies in cell physiology.