α-solanine is one of the major components of Solanum nigrum Linn., the fruits of which are used in China for food. In the present study, α-solanine was selected to assess the inhibition of arylamine N-acetyltransferase (NAT) activity and mRNA expression of NAT and kinetics in HepG2 cells. NAT activity was examined by HPLC. The double-reciprocal plot was contrived to yield a regression equation to calculate Km and Vmax. NAT mRNA expression was determined by PCR. The results revealed that α-solanine could significantly decrease NAT activity in intact HepG2 cells or the cytoplasm. Km did not differ either for intact HepG2 cells or for the cytoplasm, however Vmax was significantly different. α-solanine could decrease the expression of NAT1 mRNA and NAT2 mRNA. In summary, α-solanine was a non-competitive inhibitor of NAT in HepG2 cells. It decreased NAT activity through non-competitive inhibition of NAT activity and by decreasing the expression of NAT1 mRNA and NAT2 mRNA.