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In the present study, arsanical-based affinity chromatography for pyruvate kinase (PK) isolation was explored. p -Arsanilic acid (4-aminophenyl arsonic acid), which contains an arsonic acid moiety structurally similar to inorganic pentavalent arsenate, was conjugated to Sepharose 4B via its para -amino group to form an As(V)–Sepharose matrix. The cellular proteins from KB cells bound to arsonic acid moieties were eluted by 50 m M sodium arsenate in Tris–HCl buffer (50 m M , pH 7.6). A single protein band with a molecular mass of 58 kDa was shown on a sodium dodecyl sulfate–polyacrylamide gel. By immunoblotting, amino acid sequencing and enzymatic analysis, the sodium arsenate-eluted 58-kDa protein was demonstrated to be a human PK (type M2). By using this one-step As(V)–Sepharose chromatography, PK from KB cells was purified 35.4-fold with a specific activity of 153.15 U/mg protein in the presence of 6 m M fructose-1,6-biphosphate. Although PK was eluted from an As(V)–Sepharose column with sodium arsenate, PK activity was apparently inhibited by the used eluent system, but not by p -arsanilic acid, indicating a specific interaction of As(V) to PK. In summary, our results indicate that As(V)–Sepharose can serve as a simple and efficient chromatographic support for PK purification from KB cells.