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α-amylase inhibitors (α-AI) are a kind of active substances which show inhibitory activities to human, insect pancreas and saliva. α-AI can reduce blood glucose index in human body and kill insects, thus widely used in medicine and agriculture. This review summarizes the source, structure and action mechanisms of protein and non-protein-based α-AI, focuses on the mechanisms from the view of structure-function relationship. The applications of α-AI in disease prevention, agricultural production and plant protection are also summarized. This review may provide new insights into the comprehensive exploitation and application of α-AI from different sources.