Sulfated glycans have been found to be associated with various diseases and therefore have significant potential in molecular pathology as biomarkers. Although lectins are useful reagents for detecting glycans, there is a paucity of sulfate-recognizing lectins, and those that exist, such as from Maackia amurensis, display mixed specificities. Recombinant lectin engineering offers an emerging tool for creating novel glycan recognition by altering and/or enhancing endogenous specificities. The present study demonstrated the use of computational approaches in the engineering of a mutated form of E-selectin that displayed highly specific recognition of 6′-sulfo-sialyl Lewis X (6′-sulfo-sLe^x), with negligible binding to its endogenous nonsulfated ligand, sLe^x. This new specificity mimics that of the unrelated protein Siglec-8, for which 6′-sulfo-sLe^x is its preferred ligand. Molecular dynamics simulations and energy calculations predicted that two point mutations (E92A/E107A) would be required to stabilize binding to the sulfated oligosaccharide with E-selectin. In addition to eliminating putative repulsions between the negatively charged side chains and the sulfate moiety, the mutations also abolished favorable interactions with the endogenous ligand. Glycan microarray screening of the recombinantly expressed proteins confirmed the predicted specificity change but also identified the introduction of unexpected affinity for the unfucosylated form of 6′-sulfo-sLe^x (6′-sulfo-sLacNAc). Three key requirements were demonstrated in this case for engineering specificity for sulfated oligosaccharide: 1) removal of unfavorable interactions with the 6′-sulfate, 2) introduction of favorable interactions for the sulfate, and 3) removal of favorable interactions with the endogenous ligand.