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Kefir is a traditional dairy product with multiple probiotic characteristics derived from its associated microorganisms, including more than 50 species of lactic acid bacteria and yeast. For centuries, many people have produced kefir for human consumption; its consumption and potential role as a probiotic supplement in companion animals have never been tested. The present study explored the potential application of kefir as a probiotic supplement for dogs. Kefir was orally administered to healthy adult dogs (n = 6) for 2 wk. On d 0 and 14 (before and after kefir consumption, respectively), gut microbiota was analyzed comprehensively using quantitative PCR and 16S rDNA amplicon-based community analysis using fresh fecal samples. The 16S rDNA amplicon-based community analysis showed that the relative abundance of the phylum Fusobacteria was significantly decreased after kefir consumption. Furthermore, the relative abundance of the families Prevotellaceae, Selenomonadaceae, and Sutterellaceae increased significantly, whereas that of the families Clostridiaceae, Fusobacteriaceae, and Ruminococcaceae decreased significantly. The quantitative PCR assay showed that kefir consumption significantly increased the population of lactic acid bacteria and the lactic acid bacteria:Enterobacteriaceae ratio and significantly decreased the Firmicutes:Bacteroidetes ratio. In summary, 2-wk kefir administration successfully modified the gut microbiota without causing any clinically evident adverse effects. Therefore, kefir could be further developed as a novel probiotic food supplement for dogs to improve the quality of life of dogs.