This study was designed to examine the effects of two difference prebiotics (i.e., tart cherry and fructooligosaccharides) on cytokines and chemokines that regulate T cell homing, differentiation and activation within gut-associated lymphoid tissue of the small intestine. 8-wk-old female C57BL/6 mice were randomly assigned to treatments in a 2 x 3 factorial with antibiotics (+ or – ABX) and diet (control, tart cherry [TC] or fructooligosaccharides [FOS]) as factors. At the end of 10 wks of treatment, whole body dual-energy x-ray absorptiometry (DEXA) scans were performed, intestinal samples were collected. Fecal short chain fatty acid (SCFAs) were analyzed using gas chromatography techniques. RNA was extracted from Peyer’s patches and genes of interest were assessed using RT-PCR. Data were analyzed using 2-way ANOVA followed by post hoc testing with significant main effect or interaction was detected. FOS supplementation and not TC increased whole body BMC and BMD. The benefits of FOS on bone were unaltered in presence of antibiotics. T regulatory cells were increased within the lamina propria of the ileum with FOS and this response was suppressed with antibiotics. No changes occurred in the pro-inflammatory, Th17 cells, with FOS. Fecal SCFAs were upregulated with both TC and FOS diets, but FOS had a greater effect. Antibiotics suppressed the increase in SCFA induced by the prebiotics. Neither prebiotic increased gene expression of CCR5 and CCR9 in the Peyer’s patches, but antibiotics increased CCR9 expression. An unanticipated increase in IL-6 gene expression was noted with the TC and FOS, but the antibiotic treatment blocked this response. Although antibiotics suppressed IL-10 expression, neither TC nor FOS had an effect on this anti-inflammatory cytokine. However, both the TC and FOS suppressed the expression of the highly pro-inflammatory cytokine, IL-17. We conclude that alterations in gene expression in the Peyer’s patches with FOS supports a decrease in the IL-17 and no change in IL-10, which differs from the alterations in Th17 and Treg cell populations in the lamina propria. Furthermore, our findings indicated that FOS’s effects on bone may be mediated by some other mechanism than SCFAs’ effects on T regulatory cells via the gut-bone axis.