Significance During 150 million years of mammalian evolution, the membrane-bound mannose 6-phosphate receptor evolved high-affinity binding loops for insulin-like growth factor 2. It remains unknown whether this highly evolved ligand interaction is optimal, and whether it can be further evolved. We addressed these questions using a mutation and selection strategy that incorporated surface display and protein structure. Multiple mutations of all the binding loops were identified and improved affinity by 100-fold when combined, yet retained IGF2 specificity. Structurally, IGF2 surface interactions with binding loops were reshaped, indicating that binding site evolution could not be predicted. High IGF2 affinity binding domains could selectively inhibit IGF2-dependent cell signaling, and may be applied in therapeutic IGF2 targeting in cancer.