Previous studies have demonstrated that berberine decreases N-acetyltransferase (NAT) activity in human leukemia HL-60 cells, however, NAT gene expression has not been investigated. In this study, berberine was selected for testing the inhibition of N-acetylation of 2-aminofluorene (AF) and NAT gene expression in human HL-60 cells. The N-acetylation of AF was determined and quantitated by high performance liquid chromatography (HPLC). The data showed that a 24-hour berberine treatment decreased the amount of N-acetylation of AF in HL-60 cells. The NAT enzymes were stained and examined by Western blotting and flow cytometry. The results indicated that berberine decreased the levels of NAT protein in HL-60 cells. The expression of NAT gene (mRNAT NAT1) was determined by polymerase chain reaction (PCR), and it was found that berberine had inhibited the expression of mRNA NAT1 in human HL-60 cells.