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Stacking faults (SFs) and the interaction between solute atoms and SFs in a Mg–Bi alloy are investigated using aberration-corrected scanning transmission electron microscopy. It is found that abundant I1 SFs are generated after cold rolling and are mainly distributed inside {101¯2} twins. After aging treatment, the formation of single-layer and three-layer Bi atom segregation in the vicinity of I1 fault are clearly observed. Bi segregation also occurs at the 1/6<22¯03> bounding Frank partial dislocation cores. The segregation behaviors in I1 fault and Frank dislocations are discussed and rationalized using first-principles calculations.