Niemann−Pick type C1 (NPC1) is a large multidomain transmembrane protein essential for transporting cholesterol (CLR) from late endosomes and lysosomes to the endoplasmic reticulum and other cellular compartments. The lumen-facing Nterminal domain (NTD), involved in direct binding of CLR, is expected to have an optimum activity at acidic pH = 4.5. Here, we show that acidic pH is vital for the functionality of NPC1(NTD) and should be taken into account when studying the protein activity. We applied evolutionary, structural, and physicochemical analyses to decipher the consequences of a change in pH from acidic (pH = 4.5) to neutral (pH = 7.2) on the structural integrity of the NTD and its ability to bind CLR. We revealed that the change in pH from 4.5 to 7.2 increases the potential energy of the protein in both apo-and holo-states making the system less energetically favorable. At neutral pH, the flexibility of the protein in the apo-state is decreased caused by the alteration of specific interactions, which in turn might have a high impact on ligand recognition and binding. In contrast, neutral pH significantly exaggerates the flexibility of the protein with bound CLR that causes a partial exposure of the ligand to the water phase and its mislocation inside the ligand-binding pocket, which might obstruct CLR translocation through the membrane.