The opioid growth factor (OGF) and its receptor (OGFr) regulate human ovarian cancer cell proliferation through a tonically active inhibitory axis. We investigated the effect of OGFr overexpression on ovarian tumorigenesis. Clonal cell lines of SKOV-3 human ovarian cancer were established to stably overexpress OGFr. shRNA constructs were evaluated for antitumor activity in vitro, as well as in vivo using mouse models of subcutaneous and intraperitoneal tumor transplantation. The 5 clonal cell lines were characterized by increases in OGFr protein (62% to 245%) and binding capacity (51%-154%), and decreases (36%-185%) in cell number, relative to untransfected wild-type (WT) cells and empty vector (EV) transfected clones. Nude mice receiving subcutaneous injection of 2 overexpressing OGFr cell lines (OGFr-3 and OGFr-22) had reduced tumor incidence, delayed tumor appearance (up to 12 days), and decreased tumor volume (up to 87%) relative to WT and EV controls. Mice injected intraperitoneal with these clonal lines displayed reduced formation of tumor nodules (up to 95%), and depressed tumor weights (up to 99%) compared to WT and EV groups. DNA synthesis, but not cell survival, was depressed in cells and subcutaneous tumors overexpressing OGFr in comparison to the WT and EV groups. Angiogenesis was reduced up to 86% in clonal tumors compared to WT and EV groups. This preclinical evidence demonstrates that OGFr expression is a molecular determinant of ovarian cancer progression, and has important relevance to understanding the pathogenesis and treatment of this deadly disease.