Mammalian sterile 20-like kinase 1 (Mst1) is an ubiquitously expressed serine/threonine kinase, and its activation results in cell apoptosis. Recent studies suggest that Mst1 may function as a tumor suppressor. Here, we reported that heat shock protein 70 (Hsp70), which is thought to protect cells against cellular stress, has been identified as an Mst1-interacting protein, in a yeast two-hybrid screen of human adult prostate cDNA library with a dominant-negative Mst1 (K59R) as bait. The interaction of Mst1 with Hsp70 was confirmed by coimmunoprecipitation in both cotransfected HEK293 cells and prostate cancer cells. Hsp70 colocalized with Mst1 in the cytoplasm of LNCaP cells. The interaction sites with Mst1 consisted of NH(2)-terminal ATPase domain in Hsp70, whereas the inhibitory domain of Mst1 mediates the binding of Hsp70 in Mst1. Overexpression of Hsp70 mediates proteasomal degradation of Mst1 in a Hsp70 interacting protein (CHIP)-dependent manner. Furthermore, the proapoptotic effect of Mst1 was markedly inhibited by overexpression of Hsp70 or CHIP. Most strikingly, in response to the treatment of anticancer drug cisplatin, the induction of Hsp70 expression is higher in the androgen-independent DU145 cells compared with the androgen-dependent LNCaP cells. The higher levels of Hsp70 induction and subsequent Mst1 degradation mediate cisplatin resistance in prostate cancer DU145 cells. Moreover, overexpression of Mst1 sensitizes prostate cancer cells to cisplatin treatment. These findings implicate that Mst1, a downstream target of Hsp70, may be developed as a target for sensitizing hormone-refractory prostate cancers to chemotherapy.