The goal of this study was to examine whether supplemental fructooligosaccharides (FOS) plus mannanoligosaccharides (MOS) influenced immune function and ileal and fecal microbial populations of adult dogs. Eight adult dogs surgically fitted with ileal cannulas were used in a crossover design. Dogs were fed 200 g of a dry, extruded, kibble diet twice daily. At each feeding, dogs were dosed with either 1 g sucrose (placebo) or 2 g FOS plus 1 g MOS orally via gelatin capsule. Fecal, ileal, and blood samples were collected at the end of each 14-d period to measure microbial populations and immune characteristics. Treatment least squares means were compared using the GLM procedure of SAS. Supplementation of FOS plus MOS increased fecal bifidobacteria and fecal and ileal lactobacilli concentrations. Dogs fed FOS plus MOS also tended to have lower blood neutrophils and greater blood lymphocytes vs placebo. Serum, fecal, and ileal immunoglobulin concentrations were unchanged by treatment. Supplementation of FOS plus MOS beneficially altered indices of gut health by improving ileal and fecal microbial ecology. Supplementation of FOS plus MOS also altered immune function by causing a shift in blood immune cells.