Arsenic is a well-known human skin carcinogen whose mechanism of action remains to be elucidated. In this work using cultured human epidermal cells, arsenite suppressed accumulation of the transcriptionally active intracellular domain of Notch1. The cells responded to an active peptide from the Notch1 ligand, Jagged1, with increased levels of differentiation marker mRNAs and decreased colony-forming ability. Arsenite suppressed Jagged1 effects and expression of Jagged1 mRNA as well. Moreover, exposure of the cells to a gamma-secretase inhibitor prevented Notch1 processing, decreased cell size and differentiation marker expression, and increased proliferative potential, all effects that occur with arsenite treatment. Thus, arsenite action in suppressing keratinocyte differentiation while maintaining germinative capability could be due to inhibition of Notch1 signaling subsequent to ligand binding. This work also revealed that such arsenite action depends upon epidermal growth factor receptor kinase activity. These findings may help to explain how arsenite, by decreasing generation of the tumor suppressor Notch1, contributes to skin carcinogenesis.