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In this work, we use in-situ TEM in combination with finite elemental analysis to reveal the sub-grain boundary formation during bending of Cu single crystal. The deformation process is featured by dislocations propagation, formation of stacking faults and deformation twins, and subsequent formation of a sub-grain boundary. Finite element analysis of stress distribution indicates the stacking faults and deformation twins are all initiated from the region that corresponds to the maximum shear stress. The formation of the sub-grain boundary is through the intersection between primary and secondary stacking faults/deformation twins.