Short-chain fatty acids (SCFAs), such as butyrate, propionate, and acetate, are the major end products of bacterial fermentation of dietary fiber. SCFAs produced by intestinal bacteria have been implicated in the regulation of immune function and mucosal repair. These factors are also transferred to the placenta and affect the metabolic capacity of the fetus. The surface of the placental villi is composed of mononuclear cytotrophoblasts (CTs) and multinuclear syncytiotrophoblasts (STs) formed by differentiation and fusion of CTs. In this study, we investigate the role of SCFAs in differentiation and cell fusion. Human trophoblast cell lines, BeWo and JEG-3, were treated with acetate, propionate, and butyrate and then stimulated with dibutyl (db)-cAMP to induce cell fusion and differentiation. Treatment with butyrate or propionate increased the expression of the differentiation marker hCG alpha and beta subunits in the presence of db-cAMP. In addition, butyrate promoted the db-cAMP-induced expression of the fusion marker gene ERVFRD-1 and cell fusion. Furthermore, butyrate increased histone H3 acetylation under the differentiation stimuli. Our data suggest that butyrate promotes trophoblast cell fusion and differentiation for the formation and maintenance of placental villi during pregnancy.