The tumor suppressor gene p53 is the most frequently mutated gene found in human cancer. The majority of p53 mutations are missense mutations occurring within the DNA binding domain. Some of these mutations exhibit dominant negative effects (e.g., p53 R273H) that disrupt the normal function of wild-type p53. It is of therapeutic interest to determine if the normal function of p53 can be restored when the dominant negative effect is selectively inhibited. In this study, we tested this possibility using small hairpin RNA (shRNA) to specifically reduce the level of p53 R273H. The antisense strand of shRNA is fully complementary to mRNA of p53 R273H, but leaves a mismatch base in the middle of the duplex to wild-type p53 mRNA. Both wild-type p53 and mutant p53 R273H were transiently expressed in p53-null H1299 lung cancer cells. The shRNA we designed selectively reduced the mRNA level of p53 R273H, but had no RNA interference or antisense effects on wild-type mRNA. As a result, the transactivation activity of p53 was partially restored. In this report, we provide a new strategy for studying functional alterations for point mutation or single nucleotide polymorphism, and treating some dominant mutant-derived diseases.