The human gut microbiome, comprising bacteria, archaea, viruses, and fungi, is known to play a crucial role in health, while dysbiosis is associated with many chronic inflammatory diseases. In addition, a growing number of studies suggest that the gut microbiome might also influence bone-associated diseases such as osteoporosis. Bacterial-derived metabolites, namely short-chain fatty acids (SCFAs), are important mediators between the gut bacteria and the host and could therefore play a key role in the gut-bone axis. In addition to the known effects of SCFAs on metabolism and immune responses, the goal of our study was to examine if SCFAs influence bone health. We observed that murine as well as human osteoclast formation was reduced following treatment of osteoclast precursors with the SCFAs propionate, butyrate and valerate, with butyrate being the most potent inhibitor. Butyrate treatment also reduced the bone resorbing capacity of mature osteoclasts at concentrations >0.5mM butyrate. These inhibitory effects did not appear due to cytotoxicity, since 0.5mM butyrate had only minor inhibitory effects on cell proliferation. The effect of butyrate treatment on gene expression levels was compared at 6 hours and 24 hours. Since butyrate is known to be a histone deacetylase (HDAC) inhibitor and a ligand for G-protein-coupled receptors (GPCR) 41, 43 and 109a, our analysis is particularly focusing on pathways including genes associated with epigenetic regulation and GPCR signalling. Interestingly, GPR183, another G-protein-coupled receptor which is known to be involved in osteoclast differentiation, was one of the most downregulated genes in presence of butyrate. In conclusion, SCFAs, especially butyrate, are interesting tools to probe the mechanisms of osteoclastogenesis and may represent a novel therapeutic approach to prevent bone loss via microbial-based interventions.