Selectively 2H- and 13C-labeled spermines (SPM) were efficiently synthesized and analyzed by NMR spectroscopy to determine the spin–spin coupling constants for six conformationally relevant bonds. SPM that is composed of three alkyl moieties, a butanylene, and two propanylene chains undergoes a conformational change when interacting with multivalent anions (e.g., adenosine triphosphate (ATP), ATP–Mg2+, and tripolyphosphate). Upon interaction with ATP, the CC bonds, which affect the distance between the neighboring pairs of ammonium groups (i.e., N1/N5 and N5/N5′), increase the population of gauche rotamers by 17–20 % relative to those in the 4 HCl salt of SPM. However, the trend in increments of the gauche conformers for the SPM–ATP complex profoundly differs from that of the spermidine (SPD)–ATP complex. This implies that SPM may preferentially recognize the adenyl group of ATP rather than the tripolyphosphate moiety. This may account for the higher affinity of SPM to ATP–Mg2+ than with that of SPD, which chiefly interacts with β- and γ-phosphates and is easily replaced by Mg2+. These results may provide a clue for the further understanding of the structural basis of polyamine biological functions.