We recently discovered that the orphan G-protein-coupled receptor (GPR) 61 colocalized with GnRH receptors (GnRHRs) on the surface of most of bovine gonadotrophs. A recent study suggested that ethanolamine plasmalogen (PI) is a ligand for GPR61 in mouse neuroblastoma. Therefore, this study evaluated the hypothesis that PI alters LH and FSH secretion from cultured bovine anterior pituitary (AP) cells. We prepared bovine AP cells from postpubertal heifers (26 mo old) and cultured the cells for 3.5 d. We treated the cells with increasing concentrations (0, 5, 50, 500, 5,000, 50,000, or 500,000 pg/mL) of phosphoethanolamine PI (PEPI) extracted from the bovine brain, or l-α-lysophosphatidylethanolamine PI (LEPI) extracted from the bovine brain, for 5 min before either no treatment or GnRH stimulation. The medium samples were harvested 2 h after culture for LH and FSH assays. Phosphoethanolamine PI (50-500 pg/mL) stimulated (P < 0.05) the basal secretion of FSH but not LH. Phosphoethanolamine PI at 50 pg/mL also enhanced (P < 0.05) GnRH-induced FSH secretion. However, higher doses (500-500,000 pg/mL) of PEPI suppressed GnRH-induced FSH secretion. Moreover, 50 to 500,000 pg/mL PEPI suppressed GnRH-induced LH secretion. None of the tested concentrations of LEPI showed any effect on basal or GnRH-induced LH or FSH secretion. Pretreatment with Sma and Mad pathway inhibitors suppressed FSH secretion induced by PEPI, whereas an extracellular signal-regulated kinase pathway inhibitor blocked the PEPI-induced suppression of GnRH-stimulated LH secretion. Therefore, PEPI, but not LEPI, extracted from the bovine brain, alters FSH and LH secretion from cultured AP cells. Further studies are required to decide whether PEPI binds to GPR61 and whether PEPI plays an important role in the control of gonadotropin secretion from gonadotrophs.