Targeting the gut-bone axis with probiotics and prebiotics is considered a promising strategy to reduce the risk of osteoporosis. Gut-derived short chain fatty acids (SCFA) mediate the effects of probiotics on bone via T regulatory cells (Tregs), but it is not known whether prebiotics act through a similar mechanism. We investigated how two different prebiotics, tart cherry (TC) and fructooligosaccharide (FOS), affect bone, and whether Tregs are required for this response. Eight-week-old C57BL/6 female mice were fed diets supplemented with 10% w/w TC, FOS or a control (Con; AIN-93 M) diet and received an isotype control or CD25 antibody to suppress Tregs. FOS diet increased bone mineral content, density, and trabecular bone volume in the vertebra (~40%) and proximal tibia (~30%) compared to the TC and control diets, irrespective of CD25 treatment. Both prebiotics increased (Pâ<â0.01) fecal SCFAs, but the response was greater with FOS. To determine how FOS affected bone cells, we examined genes involved in osteoblast and osteoclast differentiation and activity as well as genes expressed by osteocytes. FOS increased the expression of regulators of osteoblast differentiation (bone morphogenetic protein 2 [Bmp2], Wnt family member 10b [Wnt10b] and Osterix [Osx]) and type 1 collagen [Col1α1]). Osteoclasts regulators were unaltered. FOS also increased the expression of genes associated with osteocytes, including phosphate regulating endopeptidase x-linked (Phex), matrix extracellular phosphoglycoprotein (Mepe), and dentin matrix acidic phosphoprotein 1 (Dmp-1). However, Sost, the gene that encodes for sclerostin was also increased by FOS as was the number and density of osteocytes. These findings demonstrate that FOS has a greater effect on bone mass and structure in young adult female mice than TC and its influence on osteoblasts and osteocytes is not dependent on Tregs.