Collagen peptides have garnered significant attention as functional foods across multiple fields due to their capacity to regulate physiological and hormonal processes, offering numerous advantages. However, despite their broad range of applications, comprehensive research on the potential toxicity of these substances remains lacking. Therefore, this study sought to assess the acute oral toxicity of a collagen peptide derived from skate (Raja kenojei) skin (CPSS) in both rats and dogs. In the rat model, CPSS was orally administered at doses of 300 and 2,000 mg/kg to Sprague-Dawley rats. An escalating single-dose oral toxicity assessment at doses of 500, 1,000, and 2,000 mg/kg was carried out in beagle dogs with 3-day intervals between doses. Throughout the 14-day post-administration assessment period, clinical signs, mortality rates, changes in body weight, and necropsy observations were closely monitored. After oral administration, no signs of toxicity associated with CPSS were observed in either rats or dogs. Therefore, the oral LD50 (approximate lethal dose for 50% mortality) for CPSS in rats was determined to exceed 5,000 mg/kg, and the maximum tolerated dose for dogs was estimated to be above 2,000 mg/kg. Consequently, this study offers safety data on the use of CPSS in functional foods and medicinal applications.