A hallmark of chronic inflammatory diseases is the excessive accumulation of white blood cells in the affected tissues, which is coordinated by pro-inflammatory mediators called chemokines. Humans have ~50 chemokines divided into two major classes – CC and CXC chemokines. Specific groups of chemokines are associated with different inflammatory diseases; for example, the chemokines CCL2, CCL7 and CCL8 are involved in atherosclerosis. As natural chemokine inhibitors, evasin proteins secreted in tick saliva are potential anti-inflammatory therapeutic agents. However, the development of tick evasins as chemokine-targeted anti-inflammatory therapeutics requires an understanding of the factors controlling their chemokine-binding specificity. Structures of the evasins EVA-P974 and EVA-AAM1001 bound to several human CC chemokine ligands (CCL7, CCL11, CCL16 and CCL17) and to a CCL8-CCL7 chimera reveal that the specificity of evasins for chemokines of the CC subfamily is defined by conserved, rigid backbone-backbone interactions. Whereas the preference for a subset of CC chemokines is controlled by side chain interactions at hotspots in flexible structural elements. The substitution of amino acid residues at hotspots provides evasin variants with new chemokine-binding properties. Moreover, we identified a novel class A evasin, EVA-AAM1001 (class A3), which possesses an additional disulfide bond near the chemokine recognition site. This additional bond allows EVA-AAM1001 to form a critical hydrophobic pocket, allowing it to bind to CC chemokines with high affinity using a different set of hotspots compared to class A1 evasins. These structural insights enabled rational engineering of evasins to tailor their chemokine selectivity. These studies provide a basis for development of evasins with applications in anti-inflammatory therapy.