The regulatory effect of the tumor-suppressor protein p53 on the apoptogenic activity of $17{alpha}$-estradiol ($17{alpha}-E_2$) was compared between HCT116 ($p53^{+/+}$) and HCT116 ($p53^{-/-}$) cells. When the HCT116 ($p53^{+/+}$) and HCT116 ($p53^{-/-}$) cells were treated with $2.5{sim}10{mu}M$ $17{alpha}-E_2$ for 48 h or with $10{mu}M$for various time periods, cytotoxicity and an apoptotic sub-$G_1$ peak were induced in the HCT116 ($p53^{+/+}$) cells in a dose- and time-dependent manner. However, the HCT116 ($p53^{-/-}$) cells were much less sensitive to the apoptotic effect of $17{alpha}-E_2$. Although $17{alpha}-E_2$ induced aberrant mitotic spindle organization and incomplete chromosome congregation at the equatorial plate, $G_2/M$ arrest was induced to a similar extent in both cell types. In addition, $17{alpha}-E_2$-induced activation of Bak and Bax, ${Delta}{Psi}m$ loss, and PARP degradation were more dominant in the HCT116 ($p53^{+/+}$) than in the HCT116 ($p53^{-/-}$) cells. In accordance with enhancement of p53 phosphorylation (Ser-15) and p53 levels, p21 and Bax levels were elevated in the HCT116 ($p53^{+/+}$) cells treated with $17{alpha}-E_2$. The HCT116 ($p53^{-/-}$) cells exhibited barely or undetectable levels of p21 and Bax, regardless of $17{alpha}-E_2$ treatment. On the other hand, although the level of Bcl-2 was slightly lower in the HCT116 ($p53^{+/+}$) than in the HCT116 ($p53^{-/-}$) cells, it remained relatively constant after the $17{alpha}-E_2$ treatment. Together, these results show that among the components of the $17{alpha}-E_2$-induced apoptotic-signaling pathway, which proceeds through mitotic spindle defects causing mitotic arrest, subsequent activation of Bak and Bax and the mitochondria-dependent caspase cascade, leading to PARP degradation, $17{alpha}-E_2$-induced activation of Bak and Bax is the upstream target of proapoptotic action of p53.