The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage-gated proton channel1, HV1 (also known as HVCN1). This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes2,3,4, histamine secretion by basophils5, sperm capacitation6, and airway pH7. The most selective ion channel known, HV1 shows no detectable permeability to other ions1. Opposing classes of selectivity mechanisms postulate that (1) a titratable amino acid residue in the permeation pathway imparts proton selectivity1,8,9,10,11, or (2) water molecules ‘frozen’ in a narrow pore conduct protons while excluding other ions12. Here we identify aspartate 112 as a crucial component of the selectivity filter of HV1. When a neutral amino acid replaced Asp 112, the mutant channel lost proton specificity and became anion-selective or did not conduct. Only the glutamate mutant remained proton-specific. Mutation of the nearby Asp 185 did not impair proton selectivity, indicating that Asp 112 has a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp 112, the mutant channel was still anion-permeable. Evidently, the proton specificity of HV1 requires an acidic group at the selectivity filter.