Depletion of fucose from human IgG1 oligosaccharide improves its affinity for Fcgamma receptor IIIa (FcgammaRIIIa). This is the first case where a glycoform modification is shown to improve glycoprotein affinity for the receptors without carbohydrate-binding capacity, suggesting a novel glyco-engineering strategy to improve ligand-receptor binding. To address the mechanisms of affinity improvement by the fucose depletion, we used isothermal titration calorimetry (ITC) and biosensor analysis with surface plasmon resonance. ITC demonstrated that IgG1-FcgammaRIIIa binding was driven by favorable binding enthalpy (DeltaH) but opposed by unfavorable binding entropy change (DeltaS). Fucose depletion from IgG1 enhanced the favorable DeltaH, leading to the increase in the binding constant of IgG1 for the receptor by a factor of 20-30. The increase in the affinity was mainly attributed to an enhanced association rate. A triple amino acid substitution in IgG1, S298A/E333A/K334A, is also known to improve IgG1 affinity for FcgammaRIIIa. ITC demonstrated that the amino acid substitution attenuated the unfavorable DeltaS resulting in a three- to fourfold increase in the binding constant. The affinity enhancement by the amino acid substitution was due to a reduced dissociation rate. These results indicate that the mechanism of affinity improvement by the fucose depletion is quite distinct from that by the amino acid substitution. Defucosylated IgG1 exhibited higher antibody-dependent cellular cytotoxicity (ADCC) than S298A/E333A/K334A-IgG1, showing a correlation between IgG1 affinity for FcgammaRIIIa and ADCC. We also examined the effect of FcgammaRIIIa polymorphism (Val158/Phe158) on IgG1-FcgammaRIIIa binding. The Phe to Val substitution increased FcgammaRIIIa affinity for IgG1 in an enthalpy-driven manner with the reduced dissociation rate. These results together highlight the distinctive functional improvement of affinity by IgG1 defucosylation and suggest that engineering of non-interfacial monosaccharides can improve glycoprotein affinity for receptors via an enthalpy-driven and association rate-assisted mechanism.