Control of heterogeneous ice nucleation (HIN) is critical for applications that range from iceophobic surfaces to ice-templated materials. HIN on 2D materials is a particular interesting topic that still lacks extensive experimental investigations. Here, we focus on the HIN on single-layer graphene (SLG) transferred onto different substrates, including silicon, silica, and thermal oxide on silicon. Complemented by other samples without SLG, we obtain a large range of wetting contact angles (WCAs) from 2° to 95°. All pristine SLG samples exhibit a large contact angle of ∼95°, which is close to the theoretical value of 96° for free-standing SLG, irrespective of the substrate and even in the presence of nanoscale wrinkles on SLG, which are due to the transfer process, indicating that the topographical features have little impact on the wetting behavior. Interestingly, SLG displays changes in hydrophobicity upon repeated water droplet freezing-melting-drying cycles due to a shift in Fermi level and/or enhanced water-substrate polar molecular interactions, likely induced by residual adsorption of H2O molecules. We found that a 0.04 eV decrease in SLG Fermi level reduces the SLG/water interface energy by ∼6 mJ/m2, thereby making SLG less hydrophobic. Counterintuitively, the reduction in SLG/water interface energy and the enhanced hydrophilicity after repeated freezing-melting-evaporation cycles actually decreases the freezing temperature by ∼3-4 °C, thereby slightly retarding rather than enhancing HIN. We also found that the water droplet freezing temperature differed by only ∼1 °C on different substrates with WCAs from 2° to 95°, an intriguing and yet reasonable result that confirms that wettability alone is not a good indicator of HIN capability. The HIN rate is rather determined by the difference between substrate/water and substrate/ice interface energies, which was found to stay almost constant for substrates weakly interacting with water/ice via van der Waals or hydrogen bonds, irrespective of hydrophilicity.