Niemann-Pick C1 like 1 (NPC1L1) is a sterol transporter expressed in the apical membrane of enterocytes and hepatocytes. NPC1L1 resembles the lysosomal NPC1 protein including an N-terminal domain (NTD), which binds a variety of sterols. The molecular mechanisms underlying this multiligand specificity of the NTD of NPC1L1 (NPC1L1-NTD) are not known. On the basis of the crystal structure of NPC1L1-NTD, we have investigated the structural details of protein-sterol interactions using molecular mechanics Poisson Boltzmann surface area calculations here. We found a good agreement between experimental and calculated binding affinities with similar ranking of various sterol ligands. We defined hydrogen bonding of sterol ligands via the 3′-β-hydroxy group inside the binding pose as instrumental in stabilizing the interaction. A leucine residue (LEU213) at the mouth of the binding pocket transiently opens to allow for the access of sterol into the binding pose. Our calculations also predict that NPC1L1-NTD binds polyene sterols, such as dehydroergosterol or cholestatrienol with high affinity, which validates their use in future experiments as close intrinsically fluorescent cholesterol analogs. A free energy decomposition and computational mutation analysis revealed that the binding of various sterols to NPC1L1-NTD depends critically on specific amino acid residues within the binding pocket. Some of these residues were previously detected as being relevant for intestinal cholesterol absorption. We show that clinically known mutations in the NPC1L1-NTD associated with lowered risk of coronary heart disease result in strongly reduced binding energies, providing a molecular explanation for the clinical phenotype.