Purpose: To study the effect of WT1 antisense oligodeoxynucleotide (ASODN) transfection on the proliferation and apoptosis of SKOV3 cells. Methods: There were four groups in our study: normal control group, WT1 ASODN group, WT1 SODN group and lipofectamine group. Cell apoptosis was observed by flow cytometry. The effect of WT1 ASODN on cell proliferation was assayed by the MTT method. RT-PCR and Western blot were used to detect the expression level of WT1 mRNA and protein. Results: The growth of the ovarian cancer cell line SKOV3 became significantly slower and its activity was reduced after being transfected by WT1 ASODN, with the inhibition rate of 49.48%. WT1 antisense phosphorothioate oligonucleotides did not only inhibit cell proliferation, arrest cell cycle at G0-G1 checkpoint and induce apoptosis in SKOV3 ovarian carcinoma cells, but also downregulated WT1 mRNA and protein expression, which contributed to the apoptosis (p < 0.05). Conclusion: WT1 antisense phosphorothioate oligonucleotides could both inhibit the proliferation and induce the apoptosis in SKOV3 ovarin carcinoma cell lines. Antisense oligonucleotides of WT1 may potentially help with the gene therapy of ovarian carcinoma.