Binding to glycosaminoglycans (GAGs) is a necessary prerequisite for the biological activity of the proinflammatory chemokine RANTES in vivo. We have applied protein engineering methods to modulate equilibrium-binding affinity as well as binding kinetics of RANTES towards its GAG ligand which also altered the chemokine’s oligomerization behavior. Out of 10 mutants, A22K and H23K were chosen for further in vitro and in vivo characterization because their stability was comparable with wild-type (wt) RANTES. In chemical cross-linking experiments, A22K gave higher and H23K lower molecular weight aggregates compared with wtRANTES as shown on SDS–PAGE. All mutants contained an N-terminal methionine residue, a well-described G-protein-coupled receptor (GPCR) antagonistic modification, which resulted in the mutants’ inability to induce monocyte chemotaxis. In surface plasmon resonance experiments using immobilized heparan sulfate (HS) and physiological buffer conditions, Met-RANTES exhibited a significantly longer residual time on the GAG chip compared with the other RANTES variants. In Scatchard plot analysis, RANTES gave a bi-phasic, bell-shaped curve suggesting ‘creation’ of ligand-binding sites on the protein during HS interaction. This was not observed in the mutants’ Scatchard plots which gave Kd values of 317.5 and 44.5 nM for the A22K and H23K mutants, respectively. The mutants were subsequently tested for their inhibitory effect in a rat model of autoimmune uveitis where only H23K exhibited a transient improvement of the clinical disease score. H23K is therefore proposed to be a GPCR-inactive GAG antagonist which displaces the wt chemokine from its natural HS-proteoglycan co-receptor. The protein engineering approach presented here opens new ways for the treatment of RANTES-related diseases.