The dried ripe seeds and pericarp of Areca catechu L., a palm species, possess significant economic value. Masticating betel nut is also a long-standing and widely prevalent lifestyle habit rooted in history, known for its stimulating effect. This effect stems primarily from arecoline, the principal active compound in betel nut. This study investigates the potential mechanisms underlying the stimulating effects of arecoline, focusing on neurotransmitters, neurotrophic factors, and the microecosystem in multi-ecological intestinal sites. After arecoline intervention in mice, significant changes were observed in locomotor activity. The levels of dopamine (DA) in liver tissue and 5-hydroxytryptamine (5-HT) in brain tissue were significantly reduced. There was a significant increase in microbial activity in the feces and in the level of n-valeric acid in the intestinal content. At the genus level, the relative abundance of Clostridium was significantly reduced, whereas the relative abundances of Helicobacter and Aquincola were markedly increased. Helicobacter, Aquincola, Faecalibaculum, and Liquorilactobacillus were signature genera in the arecoline-treated group. The 5-HT level was significantly negatively correlated with the abundance of the signature genera Aquincola, Helicobacter, and Liquorilactobacillus in the arecoline group. The ingestion of arecoline can alter the behavioral patterns of mice, causing significant changes in the 5-HT levels in brain tissue and exerting regulatory effects on the microecosystem in multi-ecological intestinal sites. These findings will provide a reference for the future development and utilization of betel nut.