The objective of this study was to investigate the effects of supplementation of fructooligosaccharides (FOS) in dry dog food on the populations of microflora, short-chain fatty acid (SCFA) production, fecal protein catabolites and fecal quality in puppies. The microbial results of the animal experiments illustrated that both 0.75% FOS and 1.00% FOS increased the amounts of Bifidobacteria and Lactobacilli, however only 1.00% FOS had the ability to reduce Escherichia coli amounts, which decreased to 4.15 lg cfu per g from 5.76 lg cfu per g of feces. SCFA results indicated that a significant increase could be made with the 0.50% FOS treatment when compared to the control. Protein catabolite results showed that 0.75% FOS decreased the fecal indole amounts significantly, to 0.29 μmol g⁻¹ from 0.36 μmol g⁻¹ of feces. Meanwhile, significant decreases of fecal phenol production were noted for the three levels of FOS. Fecal quality results showed that compared with the control, the 0.75% FOS and 1.00% FOS supplementations resulted in a significant increase of fecal scores with no significant difference between them. From the findings, it can be concluded that FOS, when used at concentrations higher than 0.75% of diet, can have a positive effect in modulating the puppy intestinal ecosystem.