Fertilization in mammals requires sperm to bind to the zona pellucida (ZP) that surrounds the egg. Galactose (Gal) or N-acetylglucosamine (GlcNAc) residues on the glycans of ZP protein 3 (ZP3) have been implicated as mouse sperm receptors. However, Mgat1(-/-) eggs with modified N-glycans lacking terminal Gal and GlcNAc residues are fertilized. To determine if Gal and GlcNAc on O-glycans of the ZP are required for fertilization, a conditional allele of the T-synthase gene (T-syn(F)) was generated. T-syn encodes core 1 beta1,3-galactosyltransferase 1 (T-synthase), which initiates the synthesis of core-1-derived O-glycans, the only O-glycans on mouse ZP3. T-syn(F/F):ZP3Cre females in which T-syn(F) was deleted at the beginning of oogenesis generated eggs lacking core-1-derived O-glycans. Nevertheless, T-syn(F/F):ZP3Cre females were fertile and their eggs bound sperm similarly to controls. In addition, T-syn(-/-) embryos generated from T-syn null eggs developed until approximately E12.5. Thus, core-1-derived O-glycans are not required for blastogenesis, implantation, or development prior to midgestation. Moreover, T-syn(-/-)Mgat1(-/-) eggs lacking complex and hybrid N-glycans as well as core-1-derived O-glycans were fertilized. The combined data show that mouse ZP3 does not require terminal Gal or GlcNAc on either N- or O-glycans for fertilization.