Acesulfame potassium (ACE-K) is an artificial sweetener widely used in many foods. This investigation assessed the cytotoxic effect of ACE-K using MTT assay in human hepatocellular carcinoma (HepG2) cell line and the genotoxic effect using chromosomal aberrations (CAs), micronucleus (MN), and comet assays in human lymphocytes. 7.5-240 μg/mL concentrations of ACE-K were applied to cells. ACE-K notably decreased the cell viability on HepG2 cells, especially at 120 and 240 μg/mL at 24 and 48 h. It also significantly reduced the mitotic index (MI) at 60, 120, and 240 μg/mL at both treatments (24 and 48 h) in human lymphocytes. The frequency of the CAs significantly increased at 60, 120, and 240 μg/mL for 48 h treatment compared to control. However, no difference was observed in the frequency of MN and nuclear division index (NDI) at all the treatments. ACE-K also induced comet tail length, tail intensity, and moment at 15 μg/mL in isolated human lymphocytes. Therefore, ACE-K showed a cytotoxic effect in HepG2 cells as well as human lymphocytes at higher concentrations. It also exhibits a mild genotoxic effect by increasing the frequency of CAs at long-term treatment and DNA damaging effect only at 15 μg/mL.