The voltage-gated proton channel (H(v)1) is a membrane protein that dissipates acute cell proton accumulations. To understand the molecular mechanisms explaining H(v)1 function, methods for purifying the protein are needed. Previously, methods were developed for expressing and purifying human H(v)1 (hH(v)1) in yeast and later in bacteria. However, these methodologies produced low protein yields and had high production costs, considerably limiting their usefulness. The protocol described in this work was developed to overcome those limitations. hH(v)1 is overexpressed in bacteria, solubilized with the detergent Anzergent 3-12, and purified by immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Our protocol produced higher protein yields at lower costs than previously published methodologies. Key features • hH(v)1, containing a poly-His tag followed by an enterokinase cutting site in its N-terminus, is overexpressed in E. coli by autoinduction. • The detergent Anzergent 3-12 is used to solubilize and purify hH(v)1 using nickel-immobilized metal affinity chromatography (IMAC). • The entire procedure can be performed in 6 days.