Wrap531α, a newly identified natural antisense transcript of p53, can regulate p53 expression upon DNA damage. We sought to investigate changes in Wrap53 and p53 levels in an osteosarcoma cell line (U-2OS) exposed to cisplatin and to study apoptosis before and after knockdown of Wrap53. Our RT-PCR analysis showed a dose-dependent 3 to 40-fold increase in Wrap53 mRNA transcript levels in U-2OS exposed to 5 to 20 μM cisplatin. An approximate 2-fold increase was also observed in transcript levels of p53 mRNA. Furthermore, transient knockdown of Wrap53 by siRNAs in U-2OS cells treated with 10 μM cisplatin reduced p53 mRNA transcript levels by up to 50% of those of controls. Immunoblotting analysis showed that in U-2OS cells treated with siRNA against exon 4 of the Wrap53 gene, the protein level of p53 was also markedly reduced. Our findings suggest that cisplatin upregulates the expression of p53 in osteosarcoma cells by upregulating the transcript levels of Wrap53. Finally, measurement of apoptotic cell death by flow cytometry showed that knockdown of Wrap53 reduced apotosis in U-2OS cells induced by cisplatin.