Core fucosylation plays a critical role in modulating the effector functions of therapeutic antibodies such as the antibody-dependent cellular cytotoxicity (ADCC) through adversely affecting the affinity of antibodies for Fcγ receptors. Thus, a facile method for Fc defucosylation of antibodies is important both for functional studies and for an enhanced therapeutic efficacy. In this chapter, we describe a detailed protocol for chemoenzymatic defucosylation of antibodies using Herceptin (trastuzumab) as a model system. The protocol includes (a) Fc deglycosylation using endoglycosidase S2 (Endo-S2); (b) enzymatic defucosylation of the resulting Fucα1,6GlcNAc-Herceptin using two distinct bacterial α-fucosidases, AlfC and BfFuc; (c) transglycosylation of the GlcNAc-Herceptin using an Endo-S2 mutant (Endo-S2 D184M) as the enzyme and a complex N-glycan oxazoline as the donor substrate; and (d) SPR analysis of the binding of antibody glycoforms with the FcγIIIA receptor. The protocol of enzymatic defucosylation of Herceptin should be equally applicable for the Fc glycan engineering of other mAbs.