We understand ¹³¹Ba as a radionuclide, which enables imaging by SPECT in nuclear medicine and provides a diagnostic match for the therapeutic alpha-emitting radionuclides ²²³Ra and ²²⁴Ra. Recently, we reported on a sufficient production route for ¹³¹Ba by irradiating a ¹³³Cs target with 27.5 MeV proton beams, and the straight-forward resin-based radiochemical separation, yielding ¹³¹Ba with high radionuclide purity. An average amount of 190 MBq of ¹³¹Ba was produced per irradiation. Apart from 0.1% isotopic impurity of ¹³³Ba, no more side-products were detectable. For the first time, radiolabeling of the complexing agent macropa (known to be an appropriate ²²⁵Ac chelator) with ¹³¹Ba was reported and mild labeling conditions as well as reaction control using TLC systems were applicable. The radiopharmacological characterization of ¹³¹Ba-labeled macropa was carried out in healthy mice using uncomplexed [¹³¹Ba]Ba²⁺ as a reference, including biodistribution studies and small animal SPECT/CT. The results revealed the rapid bone uptake of free [¹³¹Ba]Ba²⁺ ions, whereas ¹³¹Ba-labeled macropa showed a fast renal clearance and significantly lower (P < 0.001) accumulation in the bones. We therefore conclude, that ¹³¹Ba is a promising “new” radionuclide for SPECT imaging purposes and delivers appropriate quality for preclinical investigations. Moreover, the successful labeling of macropa and the in vivo stability of the ¹³¹Ba-complex are viewed as a promising starting point for the development of new heavy earth alkaline metal chelators, especially for the therapeutically relevant radium isotopes. This enables ¹³¹Ba to achieve its goal as diagnostic match and monitoring tool for ^223/224Ra.