Abstract: Apoptosis, a form of cell death, is a fundamental process for the development and maintenance of multicellular organisms that promotes the removal of damaged, senescent or unwanted cells. Induction of cancer cell apoptosis is an important strategy of anticancer therapy. In this study, we examined if melatonin, the main secretory product of the pineal gland, inhibited the growth of prostate cancer cells (LNCaP) and promoted apoptosis via mitogen-activated protein kinases (MAPKs), which are closely associated with apoptosis and survival. Melatonin treatment significantly inhibited the growth of LNCaP cells in a dose- and time-dependent manner. It clearly induced both an early stage of apoptosis (propidium iodide−, FITC Annexin-V+) and a late apoptosis/secondary necrosis (propidium iodide+ and FITC Annexin-V+), which indicated induction of serial stages of apoptosis in cells. Moreover, melatonin markedly activated c-JUN N-terminal kinase (JNK) and p38 kinase, whereas extracellular signal-regulated kinase (ERK) was not responsive to melatonin. Treatment with MAPK inhibitors, PD98059 (ERK inhibitor), SP600125 (JNK inhibitor) and SB202190 (p38 inhibitor), confirmed that melatonin-induced apoptosis was JNK- and p38-dependent, but ERK-independent. In the presence of PD98059, caspase-3 activity increased, while levels of Bax/cytochrome c (Cyt c) and Bcl-2 decreased. These effects were opposite to those observed with SP600125 and SB202190 treatments. Together, these results strongly suggest that JNK and p38 activation directly participate in apoptosis induced by melatonin. Thus, melatonin may be of promise for anti-prostate cancer strategies.