Chemoresistance is one of the leading causes that contributes to tumor relapse and poor patient outcome after several rounds of drug therapy. The causes of chemoresistance are multi-factorial. Ultimately, it is the balance of pro- and anti-apoptotic activities in the cells. We have previously reported links between POPX2 serine/threonine phosphatase with cell motility and invasiveness of breast cancer cells. Here, we show that POPX2 plays a role in the regulation of apoptosis. The effect of POPX2 on apoptosis centers on the inactivation of TGF-β activated kinase (TAK1). TAK1 is essential for several important biological functions including innate immunity, development and cell survival. We find that POPX2 interacts directly with TAK1 and is able to dephosphorylate TAK1. Cells with lower levels of POPX2 exhibit higher TAK1 activity in response to etoposide (VP-16) treatment. This subsequently leads to increased translocation of NF-κB from the cytosol to the nucleus. Consequently, NF-κB-mediated transcription of anti-apoptotic proteins is upregulated to promote cell survival. On the other hand, cells with higher levels of POPX2 are more vulnerable to apoptosis induced by etoposide. Our data demonstrate that POPX2 is a negative regulator of TAK1 signaling pathway and modulates apoptosis through the regulation of TAK1 activity. As inhibition of TAK1 has been proposed to reduce chemoresistance and increase sensitivity to chemotherapy in certain types of cancer, modulation of POPX2 levels may provide an additional avenue and consideration in fine-tuning therapeutic response.