Protein post-translational modifications play very important roles in the regulation of gene functions and successful site-directed mutagenesis is vital to determine a protein's modification sites and hence, to define its structure and function. Yet, researches are always confronted with the problem on how to generate multiple-site mutations of a target gene rapidly. In this study, a novel strategy to generate multiple-site mutations was developed based on accurate single site mutagenesis with SRrp53 as an example. Firstly, the entire SRrp53 coding sequence was amplified from the human breast cDNA library and then cloned into the expression vector pcDNA3-FLAG. Five sets of primers for K196R, K171R, K163R, K146R and K309R mutations were synthesized and were then phosphorylated at their 5' terminus. In the next step, the first run of inverse PCR was performed with one specific set of phosphorylated primers. Further, the PCR products were subjected to DpnⅠ treatment, agarose gel purification, dam methyltransferase treatment, self-ligation and purification with PCR extraction kit. Afterwards, the second run of inverse PCR was performed and the PCR products were treated as above protocol. The second processed PCR products were then used as the template for the third run of inverse PCR. The exact run times of inverse PCR are according to the number of mutation sites that you wanted. The PCR products of the last run were treated with DpnⅠ, purified with agarose gel extract kit, self-ligated and were transformed into DH5α. Ten colonies were randomly picked up for plasmids extraction and DNA sequencing. The expression of both the SRrp53 wild type and five-site mutants was analyzed by transfection of these plasmids into 293T cells. The DNA sequencing results showed that 9 of 10 extracted plasmids gained the correct mutations. And after these 9 correct plasmids were transfected into 293T cells, all of the mutants were expressed with the right molecular mass. In a word, our novel strategy can be applied to generate multiple-site mutants of a target gene efficiently and conveniently. The method has laid a good foundation for further exploration of this protein's function.