Brain-derived neurotrophic factor (BDNF)/tyrosine kinase B (TrkB) receptor signaling promotes trophoblast growth in normal and abnormal pregnancy. It also regulates the growth of malignant trophoblastic, choriocarcinoma cells. However, possible involvement of this signaling system in hydatidiform mole, another major gestational trophoblastic disease, has not been determined. Here, we found the expression of BDNF in syncytiotrophoblasts and its receptor, TrkB, in cytotrophoblasts of hydatidiform mole using real-time RT-PCR and immunoassays. In molar explant cultures, treatment with soluble TrkB ectodomain or a Trk receptor inhibitor K252a inhibited trophoblast outgrowth as well as decreased cytotrophoblast proliferation and cellular viability based on histopathological analyses and glucose metabolism monitoring. These inhibitors also increased apoptosis and caspase-3/7 activities. In an in vivo model of hydatidiform molar growth based on xenotransplantation of molar tissues into kidney capsules of SCID mice, treatment with K252a suppressed molar growth as reflected by decreased trophoblast proliferation and their invasion into mouse kidney, reduced tissue levels of chorionic gonadotropin-β, and increased apoptosis. Based on PCR array analyses to identify changes in expression profiles of cell cycle- and apoptosis-related genes in cultured molar explants, suppression of endogenous TrkB signaling led to decreases in key cell cycle-stimulatory and checkpoint genes together with the down-regulation of different antiapoptotic genes. Our findings demonstrate the importance of paracrine signaling by the BDNF/TrkB system in the proliferation and survival of molar trophoblasts. Inhibition of BDNF/TrkB signaling could provide a novel medical treatment for hydatidiform mole.