The beneficial effects of lactic acid bacteria are well known and recognized as functional foods that are health benefits for companion animals. This study, for the first time, reports the probiotic properties, safety, and whole-genome sequence ofPediococcus acidilacticiGLP06 isolated from feces of beagles. In this study, candidate probiotic bacteriaP. acidilacticiGLP02 and GLP06 were morphologically characterized and tested for their antimicrobial capacity, tolerance to different conditions (low pH, bile salts, an artificial gastrointestinal model, and high temperature), antibiotic sensitivity, hemolytic activity, cell surface hydrophobicity, autoaggregation activity, and adhesion to Caco-2 cells.P. acidilacticiGLP06 showed better probiotic potential. Therefore,P. acidilacticiGLP06 was evaluated forin vivosafety in mice and whole-genome sequencing. The results showed, that the supplemented MG06 group (1010cfu/mL), GLP06 was not only nontoxic to mice, but also promoted the development of the immune system, improved resistance to oxidative stress, and increased the diversity of intestinal microorganisms and the abundance ofLactobacillus. Whole-genome sequencing showed thatP. acidilacticiGLP06 was 2,014,515 bp and contained 1,976 coding sequences, accounting for 86.12% of the genome, with no drug resistance genes and eight CRISPR sequences. In conclusion, the newly isolated canine-derivedP. acidilacticiGLP06 had good probiotic potential, was nontoxic to mice and promoted the development of immune organs, improved the biodiversity of the intestinal flora, and had no risk of drug-resistant gene transfer, indicating thatP. acidilacticiGLP06 can be used as a potential probiotic for the prevention and treatment of gastrointestinal diseases in companion animals.