N-acetyltransferase (NAT) plays an important role in the N-acetylation of arylamine carcinogens and drugs. Shikonin has been shown to induce apoptosis and inhibit angiogenesis in vivo and in vitro. In this study, shikonin elicited dose-dependent bacteriostatic activity in Helicobacter pylori cultures. Arylamine N-acetyltransferase (NAT) activity (N-acetylation of 2-aminofluorene) was determined in the Helicobacter pylori isolated from peptic ulcer patients. Bacterial cytosols or intact cells with or without specific concentrations of shikonin co-treatment showed different percentages of 2-aminofluorene acetylation. Shikonin also inhibited N-acetylation of 2-aminofluorene in these examined Helicobacter pylori cytosols and intact cells in a dose-dependent manner. The apparent values of Km and Vmax were decreased after co-treatement with 4 μM shikonin in the cytosol of Helicobacter pylori.