Enantioselective self-assembly can be observed in some amino acid cluster ions in the gas phase. Some interesting characters have been previously observed for proline, such as the formation of homochiral dodecamers and chiral preference oscillations of small-sized clusters. However, how alkali metal ions affect the formation and stability of the related homochiral and heterochiral proline clusters is unknown. Herein, the subtle effects of sodium ions on the chiral preference of proline clusters were studied in the gas phase. For the clusters of [Pro10,11+2H]2+ with strong heterochiral preference, the substitution of one proton with one sodium ion reverses their chiral preference, making the ions of [Pro10,11+H + Na]2+ characterized by the homochiral preference. However, the second substitution of sodium ion can reverse the preference once again. The reversal of chiral preference was found to be both cluster-size and alkali metal ion sensitive. Since sodium ions are involved in a wide variety of biological processes and usually inevitably exist in laboratory experiments, the results may have an important impact on the assembly of large-sized proline-related clusters.