Bifidobacterium animalis CP‐9 was a commensal strain isolated from human breast milk. In this study, genetic and 90‐day oral toxicity were assessed in rodents for its safety. Ames test as well as in vivo bone marrow micronucleus and spermatocyte chromosomal aberration were surveyed in mice. B. animalis CP‐9 exhibited no mutagenic activity in the Ames test at the highest tested dosage (5000 µg/plate) with or without metabolic activation. No evidence of in vivo genetic toxicity was observed at the maximum tested dosage of 10 g/kg body weight (BW). Furthermore, there was no statistically significant difference of the biochemical and histological parameters in the rats administrated with B. animalis CP‐9 at dosages of 0, 0.25, 0.5, or 1.5 g/kg BW/day. No indication of concern for pathogenicity was exhibited during evaluation of Bifidobacterium ssp. generally, or B. animalis specifically. It was noted that B. animalis CP‐9 was able to survive in gastric acid‐like and high bile salt environment, and showed strong adhesion to the intestinal epithelial cells, Caco‐2. Intriguingly, B. animalis CP‐9 decreased olic acid‐induced triglyceral (TG) accumulation in the Caco‐2 cells, and viable B. animalis CP‐9 had a better bacteriostatic activity compared to another well‐documented B. animalis ssp. lactis, BB‐12. Based on the present study, B. animalis CP‐9 can be a safe probiotic supplement and may improve the health of host. Practical Application Although the health benefits of probiotics are well known, the safety of a probiotic product is acquired particularly for a long‐term consumption. We conduct the safety of B. animalis CP‐9 isolated from human breast milk, and demonstrate no toxicity concern in vitro and in vivo. Hence, B. animalis CP‐9 powder can be used as a commercial and safe probiotic supplement with some health benefits.