The Arabidopsis ATP-Binding Cassette (ABC) transporter ABCC1 sequesters arsenic (As)-phytochelatin conjugates into the vacuole, thereby conferring As resistance. Here, we report the results of a screen for phosphorylation-dependent regulation sites of AtABCC1. Variants of AtABCC1 harboring mutations that replaced amino acid residues Tyr682 , Tyr709 , Tyr822 , Ser846 , Ser1278 , or Thr1408 with alanine confer reduced resistance and decrease the intracellular As content relative to wild-type AtABCC1 when heterologously expressed in the SM7 yeast strain. This suggests that these mutations compromise the vacuolar sequestration of As by AtABCC1. Furthermore, through a phosphomimic mutant study, we found that phosphorylation of Ser846 is required for the As resistance function of AtABCC1. Our analysis provides a first clue as to the phosphorylation-mediated regulation of AtABCC1 activity.