A new polyaminocarboxylic bis-(3-hydroxy-4-pyridinone), derivative of DTPA, DTPA(PrHP)2, was synthesized starting from maltol by means of a coupling reaction of a protected 3-hydroxy-4-pyridinone and DTPA bis-anhydride. The DTPA(PrHP)2 acid-base behavior was investigated by UV–Vis spectrophotometry and spectrofluorimetry at I = 0.15 mol L−1 in NaCl(aq) at T = 298.15 K and T = 310.15 K. To confirm the speciation scheme, also 1H NMR measurements were performed at T = 298.15 K. The protonation constants obtained from the cited different analytical techniques showed good accordance and were also in agreement with data reported in the literature. The complexing ability of DTPA(PrHP)2 towards divalent (Ca2+, Cu2+, Zn2+) and trivalent (Al3+, Fe3+) cations was studied by potentiometric, UV–Vis spectrophotometric and 1H NMR titrations at T = 298.15 K and I = 0.15 mol L−1 in NaCl(aq). The fitting analysis of experimental data led to the determination of different speciation models consisting of MpLqHr(pn+r-5q) species with different stoichiometry and protonation stages, hydrolytic mixed and polynuclear complexes. The equilibrium model and complex formation constants, obtained from various analytical techniques, are in good agreement. The sequestering ability of the ligand towards the metal cations was assessed through the determination of the pL0.5 and pM parameters at different pH values and pH = 7.4, respectively, which showed to follow the trend Fe3+ > Al3+ > Cu2+ > Ca2+ > Zn2+. Finally, biodistribution studies were carried out in mice previously injected with the radiotracer 67Ga–citrate to evaluate the in vivo ability of DTPA(PrHP)2 as chelating agent towards trivalent metal cations.