Human insulin (HI) has fascinating metal-facilitated self-assembly properties that are essential for its biological function. HI has a natural Zn²⁺ binding site and we have previously shown that covalently attached abiotic ligands (e.g., bipyridine, terpyridine) can lead to the formation of nanosized oligomeric structures through the coordination of metal ions. Here we studied the hypothesis that metal ions can be used to directly control the pharmacokinetics of insulin after covalent attachment of an abiotic ligand that binds metal ions. We evaluated the pharmacokinetics (PK) and biodistribution of HI self-assemblies directed by metal ion coordination (i.e., Fe²⁺/Zn²⁺, Eu³⁺/Zn²⁺, Fe²⁺/Co³⁺) using preclinical SPECT/CT imaging and ex vivo gamma counting. HI was site-specifically modified with terpyridine (Tpy) at the Phe^B1 or Lys^B29 position to create conjugates that bind either Fe²⁺ or Eu³⁺, while its natural binding site (His^B10) preferentially coordinates with either Zn²⁺ or Co³⁺. HI was also functionalized with trans-cyclooctene (TCO) opposite to Tpy at Phe^B1 or Lys^B29, respectively, to allow for tetrazine-TCO coupling via a tetrazine-modified DTPA followed by ¹¹¹In-radiolabeling for SPECT/CT imaging. When the ¹¹¹In-B29Tpy-HI conjugate was coordinated with Fe²⁺/Zn²⁺, its retention at the injection site 6 h after injection was ~8-fold higher than the control without the metal ions, while its kidney accumulation was lower. ¹¹¹In-B1Tpy-HI showed comparable retention at the injection site 6 h after injection and slightly increased retention at 24 h. However, higher kidney accumulation and residence time of degraded ¹¹¹In-B1Tpy-HI was observed compared to that of ¹¹¹In-B29Tpy-HI. Quantitative PK analysis based on SPECT/CT images confirmed slower distribution from the injection site of the HI-metal ion assemblies compared to control HI conjugates. Our results show that the Tpy-binding site (i.e., Phe^B1 or Lys^B29) on HI and its coordination with the added metal ions (i.e., Fe²⁺/Zn²⁺ or Fe²⁺/Co³⁺) directed the distribution half-life of HI significantly. This clearly indicates that the PK of insulin can be controlled by complexation with different metal ions.