Functionalization　of　graphene　enables　precise　control　over　interlayer　spacing　during　film　formation,　thereby　enhancing　the　separation　efficiency　of　radioactive　ions　in　graphene　membranes.　However,　the　systematic　impact　of　interlayer　spacing　of　graphene　membranes　on　radioactive-ion　separation　remains　unexplored.　This　study　aims　to　elucidate　how　interlayer　spacing　in　functionalized　graphene　membranes　affects　the　separation　of　radioactive　ions.　Utilizing　polyamidoxime　(PAO)　to　modify　graphene　oxide,　we　controlled　the　interlayer　spacing　of　graphene　membranes.　Experimental　results　indicate　that　tuning　interlayer　spacing　enables　control　of　the　permeation　flux　of　radioactive　ions　(UO22+　1.01　×　10−5–8.32　×　10−5　mol/m2·h,　and　K+　remains　stable　at　3.60　×　10−4　mol/m2·h),　and　the　K+/UO22+　separation　factors　up　to　36.2　at　an　interlayer　spacing　of　8.8　Å.　Using　density　functional　theory　and　molecular　dynamics　simulations,　we　discovered　that　the　effective　separation　is　mainly　determined　via　interlayer　spacing　and　the　quantity　of　introduced　functional　groups,　explaining　the　anomalous　high　permeation　flux　of　target　ions　at　low　interlayer　spacing　(4.3　Å).　This　study　deepens　our　comprehension　of　interlayer　spacing　within　nanoconfined　spaces　for　ion　separation　and　recovery　via　graphene　membranes,　offering　valuable　insights　for　the　design　and　synthesis　of　high-performance　nanomembrane　materials.　©　2024　Elsevier　B.V.