objective: Chronic heavy drinking causes ethanol-induced osteoporosis (EIO). The present study aimed to explore the role of GM in EIO. methods: A rat EIO model was established by chronic ethanol intake. Taking the antibiotic application as the matched group of dysbacteriosis, an integrated 16S rRNA sequencing and liquid chromatography-tandem mass spectrometry-based metabolomics in serum and faeces were applied to explore the association of differential metabolic phenotypes and screen out the candidate metabolites detrimental to ossification. The colon organoids were used to track the source of 5-HT and the effect of 5-HT on bone formation was examined in vitro. results: Compared with antibiotics application, ethanol-gavaged decreased the BMD in rats. We found that both ethanol and antibiotic intake affected the composition of GM, but ethanol intake increased the ratio of Firmicutes to Bacteroidetes. Elevated serotonin was proved to be positively correlated with the changes of the composition of GM and faecal metabolites and inhibited the proliferation and mineralization of osteogenesis-related cells. However, the direct secretory promotion of serotonin was absent in the colon organoids exposed to ethanol. conclusions: This study demonstrated that ethanol consumption led to osteoporosis and intestinal-specific dysbacteriosis. Conjoint analysis of the genetic profiles of GM and metabolic phenotypes in serum and faeces allowed us to understand the endogenous metabolite, 5-HT, as detrimental regulators in the gut-bone axis to impair bone formation.