Abstract: Prostaglandins (PGs) are profound hormones in teleost sexual behavior, especially in mating. PGs act as pheromones that affect the olfactory sensory neurons of males, inducing the initiation of a series of mating behaviors. However, the molecular mechanism by which PGs trigger mating behavior in ovoviviparous teleosts is still unclear. In the present study, we employed the ovoviviparous black rockfish (Sebastes schlegelii), an economically important marine species whose reproductive production is limited by incomplete fertilization, as a model species. The results showed that when the dose of PGE₂ was higher than 10 nmol/L, a significant (P < 0.05) increase in mating behaviors was observed. Dual-fluorescence in situ hybridization indicated that PGE₂ could fire specific neurons in different brain regions and receptor cells in the olfactory sac. After combining with specific neurons in the central nervous system (CNS), a series of genes related to reproduction are activated. The intracerebroventricular administration of PGE₂ significantly increased lhb levels (P < 0.05) in both sexes. Moreover, steroidogenesis in gonads was also affected, inducing an increase (P < 0.05) in E₂ levels in males and T levels in females. PGE₂ levels were also increased significantly (P < 0.05) in both sexes. The present study revealed that PGE₂ can activate mating behavior in black rockfish in both hormone and pheromone pathways, leading to variations in sex steroid levels and activation of reproductive behaviors. Our results provide not only novel insight into the onset of mating behaviors in ovoviviparous teleosts but also solutions for the incomplete fertilization caused by natural mating in cage aquaculture.