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The surging market demand for sea cucumbers (Apostichopus japonicus) has fueled a thriving growth in the sea cucumber breeding industry. Pheromones are molecules that evolve signals in defined proportions in the case of multicomponent pheromones, emitted by one individual and received by a second individual of the same species to cause a specific response. It remains an enigma as to how the species of sea cucumber, devoid of visual, auditory, and olfactory senses, aggregate and communicate within their groups, as well as coordinate gamete release during the breeding period. We hypothesize that pheromones may serve as the medium for facilitating these behaviors and propose conducting experiments centered around pheromonal investigation. In this study, we aimed to analyze common metabolites of male A. japonicususing LC-MS/MS technology and bioinformatics. Coelomic fluid and ejaculate fluid were analyzed, with aquaculture water serving as the control. Eight target pheromones were analyzed, and eight of metabolite analogs were tested for aggregation and spawning behaviors. Results showed that β-sitosterol at a concentration of 10 μg·L−1significantly promoted aggregation (21.25 % average rate) and induced a significant increase in spawning rate (58 %) of A. japonicus. The significance of this study lies in the fact that by collecting and utilizing pheromones of A. japonicus, farmers can accurately determine the reproductive cycle and optimal breeding season for A. japonicus. This enables fish farmers to implement effective breeding management strategies at the appropriate time, thereby increasing the success rate of reproduction and providing valuable technical support for the cultivation of A. japonicusseedlings.