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To investigate the microstructure-property relations of honeybee stingers, the cross-section microstructures were analyzed by scanning electron microscope (SEM) and the mechanical properties of honeybee stingers were tested by nanoindentation experiment in vivo in this paper. The Young’s modulus and hardness in the cross section of different segments of honeybee stingers were obtained. It is found that the honeybee stinger is of a hierarchical structure in cross section, which varies from the root to the tip and leads to quite different mechanical properties of the stingers. The natural optimized microstructure and excellent mechanical properties of the stingers effectively contribute to the biological function and self-protection performance of honeybees.