The role of matrix metalloproteinases (MMPs) in endometriosis, a gynecological disease of women, is unclear. The study investigated the activity of MMP-3 and its interplay with MMP-9 during the onset of endometriosis. Additionally, the importance of MMP-3 on the apoptotic pathway in endometriosis and effect of melatonin thereon were investigated. A Significant increase in the activity of MMP-3 with the severity of endometriosis in human was observed which was found similar in mice also. During the early phase of endometriosis, MMP-3 but not MMP-9 was increased and associated with the expression of transcription factor, c-Fos. Moreover, urokinase plasminogen activator and tissue inhibitor of metalloproteinase (TIMP)-3 were involved in MMP-3 regulation during endometriosis. Furthermore, MMP-3 activity that was parallel to c-Fos expression in endometriosis was reduced by melatonin pretreatment as characterized by diminished activator protein (AP)-1 DNA-binding activity. Because decreased apoptosis is an explanation for the perpetuation of endometriosis, we tested the role of melatonin on apoptotic pathway in preventing endometriosis. Significant regression of glandular epithelium was observed in melatonin-treated when compared to untreated mice. Melatonin treatment increased apoptotic cells in endometriotic zones. This was related to reduced Bcl-2 expression along with increased Bax expression and caspase-9 activation. In summary, early induction of MMP-3 was distinct from MMP-9 during endometriosis, which was regulated by c-Fos and TIMP-3. Melatonin suppressed MMP-3 activity and amplified apoptosis while regressing endometriosis through a caspase-3 mediated pathway. Thus, melatonin may be a therapeutic agent for resolving endometriosis.