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Background/objectives: Obesity, characterized by abnormal fat accumulation and metabolic disturbances, presents a significant health challenge. Opuntia humifusa Raf., commonly known as Korean Cheonnyuncho, is rich in various beneficial compounds and has demonstrated antioxidant and anti-inflammatory effects. However, its potential impact on glucose and lipid metabolism, particularly in obese rats, remains unexplored. We aimed to investigate whether O. humifusa stems and fruits could beneficially alter glucose metabolism and lipid profiles in a rat model of high-fat diet (HFD)-induced obesity., Materials/methods: Thirty-two rats were allocated into 4 groups: normal diet (NF), HFD control (HF), HFD treated with 2% O. humifusa stems (HF-OS), and HFD treated with 2% O. humifusa fruits (HF-OF). Experimental diets were administered for 6 weeks. At the end of the treatment, liver and fat tissues were isolated, and serum was collected for biochemical analysis. The major flavonoid from O. humifusa stems and fruits was identified and quantified., Results: After 6 weeks of treatment, the serum fasting glucose concentration in the HF-OS group was significantly lower than that in the HF group. Serum fasting insulin concentrations in both HF-OS and HF-OF groups tended to be lower than those in the HF group, indicating a significant improvement in insulin sensitivity in the HF-OS group. Additionally, the HF-OS group exhibited a tendency towards the restoration of adiponectin levels to that of the NF group., Conclusion: The 2% O. humifusa stems contain abundant quercetin and isorhamnetin, which alter fasting blood glucose levels in rats fed a HFD, leading to a favorable improvement in insulin resistance., Competing Interests: Conflict of Interest: The authors declare no potential conflicts of interests., (©2024 The Korean Nutrition Society and the Korean Society of Community Nutrition.)