Serotonin is a well-known secondary metabolite that plays an important role in many growth and developmental processes, as well as biotic and abiotic stress responses of plants. Yet, whether serotonin biosynthesis regulates the resistance of rice to the brown planthopper (BPH) Nilaparvata lugens and its underling mechanisms have not been entirely investigated. In this study, we found that expression levels of four serotonin biosynthesis gene, OsTDC1, OsTDC2, OsTDC3, and OsT5H, and the serotonin content were significantly induced by BPH nymph infestation. The rice seedlings pretreated with serotonin were more attractive to BPH nymphs and promoted their feeding behavior as indicated by increased honeydew excretion. In addition, serotonin application to artificial diets increased the survival rates of BPH nymphs in a dose-dependent manner compared to the controls. Moreover, the exogenous serotonin application to rice seedlings increased the average injury scale and functional plant loss indices caused by BPH nymph infestation. Moreover, supplemented serotonin enhanced soluble sugar and free amino acid contents, as well as the activities of SOD, POD, and PPO, but repressed the production of flavonoids. Our findings illustrated that serotonin plays a negative role in the regulation of rice resistance to BPH nymphs probably by modulating contents of soluble sugars, free amino acids, and flavonoids.