The opioid growth factor (OGF) and its receptor, OGFr, serve as a tonically active inhibitory axis regulating the proliferation of human ovarian cancer cells. In the present study, we have investigated the repercussion on the progression of this deadly neoplasia when cells are engineered to molecularly under-express OGFr. shRNA constructs were used to knockdown OGFr in SKOV-3 cells; two clonal cell lines were examined. OGFr protein expression was decreased up to 73% in clones compared with wild-type (WT) and empty vector (EV) controls. OGFr-binding assays of clones revealed 50-55% decreases in binding capacity compared with control cells; binding affinity was comparable in all groups. Cell number in clones was increased 33-132%, and doubling times decreased 29-35%, compared with WT and EV cultures. Addition of exogenous OGF or naltrexone did not affect cell number in cultures with silenced OGFr. DNA synthesis of clonal cell lines was increased 136-146% from the WT and EV groups; no changes were noted in cell survival. Nude mice injected subcutaneously with cells under-expressing OGFr had an increased tumor incidence, decreased latency to tumor formation, increased tumor volume and decreased OGFr expression in tumors compared with WT and EV controls. OGF treatment in mice with WT or EV tumors, but not OGFr under-expressing tumors, inhibited tumor volume and weight. Collectively, these data demonstrate the critical nature of the OGF-OGFr axis as a determinant of the progression of human ovarian cancer, and suggest that attenuation of this system has an important bearing on the survival of these patients.