Inversely, PP2A-mediated dephosphorylation of Bax at S184 enhances the Talazoparib proapoptotic function of Bax. Another phosphorylation site of Bax is S163 that has been shown to be phosphorylated by GSK-3b in vitro and in vivo, which is associated with promotion of apoptosis in neuronal cells. Moreover, it has recently been demonstrated that Bax can also be phosphorylated at threonine167 site by stress-activated JNK and/or p38 kinase, which leads to Bax activation and mitochondrial localization prior to apoptosis. The proapoptotic activity of Bax can be regulated by phosphorylation but the molecular mechanism involved is not fully understood. The purpose of this study is to investigate the mechanism of how phosphorylation status of Bax at distinct site affects the Bax-mediated mitochondrial cell death pathway. Here we chose the site-directed mutagenesis approach to generate the non-phosphorylatable and the phosphomimetic Bax mutants at different site. Results indicate that the S184 phosphorylation site plays a more important role than the S163 phosphorylation site in regulating the proapoptotic function of Bax. The Bcl2 family lies at the central of the intrinsic pathway to apoptosis, the interactions between the family members being critical for determining the fate of a cell. The multi-domain pro-apoptotic members, including Bax and Bak, have been demonstrated that one or other of these proteins are required for apoptotic cell death through the release of apoptogens from mitochondria. However, the mechanism by which the cell death mediator Bax becomes activated to cause mitochondrial damage remains elusive. Mounting evidence indicates that the proapoptotic function of Bax can be regulated by phosphorylation at various sites through activation of various protein kinases, including PI3K/AKT, GSK-3b, JNK and p38 kinases. Intriguingly, phosphorylation at S163 and S184 has been reported to play an inverse role in regulating the proapoptotic function of Bax. To flesh out the functional contribution of individual phosphorylation site, we chose to abrogate Bax phosphorylation by introducing a conserved, non-phosphorylated alanine at the S163 or S184 phosphorylation site. Similar mutant carrying a glutamate at these same sites were prepared in order to functionally mimic the charge conferred by the phosphate. The power of this genetic approach is intensified in these studies because a direct comparison can be made between the nonphosphorylatable and phosphomimetic Bax mutants expressed at the same levels in the same cell types. We believe that this comparative approach can provide a definitive answer to explain how, and at which site phosphorylation affects the proapoptotic function of Bax. Results clearly indicate that the non-phosphorylatable S184Acontaining Bax mutants function as active forms of Bax, which exhibit the highest cell killing activity. Inversely, the phosphomimetic S184E-containing mutants almost lost their proapoptotic activities. These findings support and extend our and others’ previous studies that phosphorylation of Bax at S184.