Diallyl sulfide (DAS) is one of the major components of garlic (Allium sativum) and is widely used in the world for food. In this study, DAS was selected for testing the inhibition of arylamine N-acetyltransferase (NAT) activity (N-acetylation of 2-aminofluorene) and gene expression (mRNA NAT) in human colon cancer cell lines (colo 205, colo 320 DM and colo 320 HSR). The NAT activity was examined by high performance liquid chromatography and indicated that a 24 h DAS treatment decreases N-acetylation of 2-aminofluorene in three colon (colo 205, 320 DM and colo 320 HSR) cancer cell lines. The NAT enzymes (protein) were analyzed by western blotting and flow cytometry and it indicated that DAS decreased the levels of NAT in three colon (colo 205, 320 DM and colo 320 HSR) cancer cell lines. The gene expression of NAT (mRNAT NAT) was determined by polymerase chain reaction (PCR), it was shown that DAS affect mRNA NAT expression in examined human colon cancer cell lines. This report is the first to demonstrate that DAS does inhibit human colon cancer cell NAT activity and gene expression.