Biologically active conformations of the IgG1 Fc homodimer are maintained by multiple hydrophobic interactions between the protein surface and the N-glycan. The Fc glycan modulates biological effector functions, including antibody-dependent cellular cytotoxicity (ADCC) which is mediated in part through the activatory Fc receptor, Fc gamma RIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein-carbohydrate interface (F241A, F243A, V262E, and V264E) increased galactosylation and sialylation of the Fc and, concomitantly, reduced the affinity for Fc gamma RIIIA. We rationalized this effect by crystallographic analysis of the IgG1 Fc F241A mutant, determined here to a resolution of 1.9 angstrom, which revealed localized destabilization of this glycan-protein interface. Given that sialylation of Fc glycans decreases ADCC, one explanation for the effect of these mutants on Fc gamma RIIIA binding is their increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated and hypersialylated glycans exhibited unchanged binding affinity to Fc gamma RIIIA. Moreover, when we expressed these mutants as a chemically uniform (Man(5)GlcNAc(2)) glycofonn, the individual effect of each mutation on Fc gamma RIIIA affinity was preserved. This effect was broadly recapitulated for other Fc receptors (Fc gamma RI, Fc gamma RIIA, Fc gamma RIIB, and Fc gamma RIIIB). These data indicate that destabilization of the glycan protein interactions, rather than increased galactosylation and sialylation, modifies the Fc conformation(s) relevant for Fc gamma R binding. Engineering of the protein-carbohydrate interface thus provides an independent parameter in the engineering of Fc effector functions and a route to the synthesis of new classes of Fc domain with novel combinations of affinities for activatory and inhibitory Fc receptors.