Traditional medicine and herbal remedies are gaining popularity worldwide, comprising a significant portion of healthcare research, advancements, and market demand. Growing scientific evidence supports their substantial efficacy as pharmaceutical ingredients and dietary supplements in preventive healthcare. When developing pharmaceuticals, it is crucial to ensure that ingredients are free from side effects and toxicity in order to prioritize safety. Geckos, known as shou gong, are a diverse group of lizards that are widely utilized for treating various diseases in Korean Medicine. This study was conducted to assess the potential acute toxicity of a water extract Gekko gecko by a single oral dose in Sprague-Dawley rats. Twenty rats of each sex were randomly assigned to four groups (5 rats each). Test articles were administrated once by oral gavage to rats at dose levels of 0, 500, 1,000, or 2,000 mg/kg body weight. Mortality, changes of body weight, and clinical signs of gross observation were monitored for 14 days after dosing. At the end of a 14-day observation period, all animals were sacrificed and complete macroscopic and hematological examinations were performed. There was no dead animal or test article-related effect on clinical signs, body weight, or gross finding. Other specific changes were not found between control and treated groups in hematology. Results showed no adverse effect at a dose of 500, 1,000, or 2,000 mg/kg in rats. The minimal lethal dose was considered to be over 2,000 mg/kg body weight in rats.