Tanshinone IIA (Tan IIA), as a bioactive compound extracted from the dried roots of Salvia miltiorrhiza (also known as Danshen), is known to inhibit cancer cell proliferation and induce apoptosis. However, the mechanisms underlying the function of Tan IIA in cancer cell apoptosis remain to be elucidated The aim of the present study was to identify the molecular mechanisms underlying the anti-cancer effects of Tan IIA in p53-deficient H1299 cells. Tan IIA was demonstrated to suppress murine double minute 4 (MDM4) expression in a time- and dose-dependent manner through the inhibition of MDM4 mRNA synthesis. Tan IIA-induced downregulation of MDM4 resulted in an increase of P73 alpha and a decrease of inhibitor of apoptosis 3 (IAP3). However, P73 alpha was not activated as two P73 alpha target genes, BCL2 binding component 3 and phorbol-12-myristate-13-acetate-induced protein 1, were not significantly induced. Tan IIA-induced inhibition of IAP3 expression may be involved in Tan IIA-induced apoptosis and inhibition of H1299 cell viability. Notably, a combination of Tan IIA and doxorubicin (DOX) exposure resulted in further MDM4 overexpression in H1299 cells, indicating that Tan IIA sensitized p53-deficient and MDM4-overexpressing H1299 cells to DOX-induced apoptosis.