The mechanism of ice nucleation at the molecular level remains largely unknown. Nature endows antifreeze proteins (AFPs) with the unique capability of controlling ice formation. However, the effect of AFPs on ice nucleation has been under debate. Here we report the observation of both depression and promotion effects of AFPs on ice nucleation via selectively binding the ice-binding face (IBF) and the non-ice-binding face (NIBF) of AFPs to solid substrates. Freezing temperature and delay time assays show that ice nucleation is depressed with the NIBF exposed to liquid water, whereas ice nucleation is facilitated with the IBF exposed to liquid water. The generality of this Janus effect is verified by investigating three representative AFPs. Molecular dynamics simulation analysis shows that the Janus effect can be established by the distinct structures of the hydration layer around IBF and NIBF. Our work greatly enhances the understanding of the mechanism of AFPs at the molecular level and brings insights to the fundamentals of heterogeneous ice nucleation.antifreeze proteins | ice nucleation | Janus effect | interfacial water | selective tethering A ntifreeze proteins (AFPs) protect a broad range of organisms inhabiting subzero environments. The function of AFPs lies in lowering the freezing point in a noncolligative manner (1, 2). It has been shown that AFPs can adsorb on the ice crystal surface with the ice-binding face (IBF, also termed ice-binding site or icebinding surface) (3, 4). The adsorbed AFPs lead to curvatures on the ice surface between adjacent AFPs, and ice growth is retarded due to the Kelvin effect, which is known as the adsorptioninhibition mechanism (5). However, whether the non-ice-binding face (NIBF) is involved in the function of AFPs and what effect the NIBF exerts are rarely studied (4, 6, 7). On the other hand, the effect of AFPs on ice nucleation (8) is still under intense debate (9-13), although ice nucleation, the formation of a stable nucleus with a critical size, is the control step for ice formation (8). Liu et al. (9) suggested that AFPs could inhibit heterogeneous ice nucleation of water, whereas research on ice nucleation of microdroplets of AFP solutions exhibited no obvious effect of AFPs in inhibiting ice nucleation (10). It was also reported that an AFP solution with high concentration facilitated ice nucleation (11). The contradiction also exists for the research of ice nucleation on solid surfaces immobilized with AFPs (12, 13). Therefore, it is highly desirable to elucidate the exact role of AFPs on ice nucleation and to correlate AFP structures at the molecular level with their function in tuning ice nucleation, which is essential for practical applications in food, pharmaceutical, and chemical industries (14,15).Herein, we investigate the effect of IBF and NIBF of AFPs on ice nucleation via binding AFPs to solid substrates in a way that either IBF or NIBF is exposed to liquid water. This binding method is readily extendable to other AFPs because of the clear distinction...