Arsenic trioxide has been used as a therapeutic agent for many diseases and has good clinical efficacy. However, arsenic resistance in many cells limits its clinical application, and the mechanism is not fully determined. To further understand the mechanism of arsenic resistance, we constructed human arsenic-resistant ECV-304 cells (AsRE) and identified the arsenic resistant related gene ABCA1, which belonged to ATP-binding cassette subfamily in the previous study. In this study, we found that when ABCA1 expression was silenced, the AsRE cells lost their arsenic tolerance, and arsenic accumulation was greater than that of the parental ABCA1-bearing counterparts. Conversely, overexpression of ABCA1 in Hela cells decreased arsenic accumulation, and the cells were more resistant to As(III) than the control that was transfected with empty vector. Our results suggest a novel role for ABCA1 in the development of mammalian arsenic resistance.