Objective To validate the feasibility of genotoxicity assessment of food ingredients using high-content screening (HCS) in vitro micronucleus (IVMN) assays. Methods Two IVMN methods were used to evaluate the genotoxicity of ten compounds, including five known genotoxic compounds (clastogens and aneugens), one known non-genotoxic compound and four food ingredients. At least three concentrations of each compound and two parallels were set. No serum minimum essential medium(MEM)was treated as negative control. 20 μg/ml cyclophosphamide and 1.0 μg/ml mitomycin C was treated as positive controls. With and without metabolic activation, Chinese hamster lung(CHL)cells were treated with ten test compounds for 4 h. And the frequency of micronuclei was analyzed. Results The frequencies of micronuclei, which were obtained by conventional IVMN and HCS IVMN assay, induced by benzo(a)pyrene (dose range from 2.5 to 10 μg/ml), methyl methanesulphonate (dose range from 5 to 20 μg/ml), 4-nitroquinoline-N-oxide (dose range from 0.01 to 0.04 μg/ml), colchicine (dose range from 0.25 to 1.0 μg/ml), and vinblastine sulfate (dose range from 0.5 to 2.0 μg/ml) were significantly different with negative controls(P<0.05), and there was a obvious dose-response relationship. The frequencies of micronuclei induced by sodium chloride (dose range from 1 250 to 5 000 μg/ml), food ingredient A (dose range from 1 250 to 5 000 μg/ml), food ingredient B (dose range from 1 250 to 5 000 μg/ml), food ingredient C (dose range from 312.5 to 1 250 μg/ml), and food ingredient D (dose range from 156.25 to 625 μg/ml) showed a gradual upward trend in dose-response curve. Compared with negative controls, the differences of the result were not statistically significant(P>0.05). Conclusion In this study, the result of HCS IVMN assays were accordance to that of traditional IVMN assay. It was revealed that the genotoxicity assessment of food ingredients using HCS IVMN method was feasible.