AbstractAntifreeze glycoprotein (AFGP) is an O‐glycoprotein that displays antifreeze activity through depression of the freezing point of water. GalNAc is a core sugar structure of AFGP, and contributes to induce antifreeze activity of this glycoprotein. However, the general functional role that this sugar plays at the molecular level is still unknown. To elucidate this, it is essential to determine the relationship between structure and activity of O‐GalNAcylated AFGP using homogeneous glycoproteins. Thus, the total synthesis of homogeneous O‐GalNAcylated AFGP was conducted by using a unique peptide derivative: peptidyl‐N‐pivaloylguanidine. It was found that peptidyl‐N‐pivaloylguanidine is an “unreactive” peptide in peptide coupling reactions but is interconvertible with a “reactive” peptide‐α‐thioester by means of a simple treatment under buffer condition at pH=7 to 8. The unique switchable reactivity of peptidyl‐N‐pivaloylguanidine enabled an efficient sequential peptide coupling strategy. By using this strategy, various lengths of homogeneous O‐GalNAcylated AFGP were synthesized, including one that was 120 amino acids in length, with 40 O‐GalNAcylation sites. The structural analysis by circular dichroism spectroscopy and evaluation of the antifreeze activity of the synthetic AFGP(GalNAc)s revealed that the simple O‐glycosylation with GalNAc is essential for both structural and functional basis of AFGP to exhibit antifreeze activity.