We report on the disparity in the assembly behavior of four types of nano‐sized macroions induced by isotopic substitution of protium (H) to deuterium (D) in solvents. Macroions with modest charge density can self‐assemble into single‐layer, hollow, spherical “blackberry”‐type structures, with larger assembly sizes representing stronger attractions among the macroions. Kinetically, all assembly processes become slower in D2O than in H2O. Thermodynamically, the polyoxometalate {SrPd12}, the uranium cage {U60} with alkali metal counterions, and the metal–organic cationic cage {Pd12L24} demonstrate similar assembly sizes in both H2O and D2O, whereas the metal oxide cluster {Mo72Fe30} as a weak acid shows an unusually large assembly size in H2O—suggesting a stronger contribution from the hydrogen bonding in the last case.