The antifreeze mechanism of antifreeze glycoproteins (AFGPs) remains incompletely understood, which limits the design of new antifreeze molecules for practical applications. For instance, the ice growth inhibition of AFGP8 (the shortest AFGPs) is primarily driven by hydrophobic methyl and hydrogen-bonding hydroxyl groups. However, altering the C3-β linkage in the disaccharide moiety of AFGP8, denoted as variant GP8-LacNAc, significantly reduces its antifreeze activity. This challenges the conventional understanding of the antifreeze activity of AFGP8 because no group is removed. Here, we revisit the antifreeze mechanism using molecular dynamics simulations of two AFGPs as an example, revealing the relation between conformation, ice-binding group, and ice growth inhibition activity. The PPII helix is not the reason for the weak activity of GP8-LacNAc, while the cooperativity of the conformation and group matters. AFGP8 maintains a dominant conformation with all four disaccharides on the same side, which maximizes the binding with ice. While GP8-LacNAc undergoes frequent conformational transitions, leading to a methyl-hydroxyl distance mismatch with the ice surface. Our results reveal the cooperative role of conformation and ice-binding groups, providing new insights into the antifreeze mechanism of flexible proteins.