Multidrug resistance protein AtMRPs belong to the ATP binding cassette (ABC) transporter super family. ABC proteins are membrane proteins involved in the transport of a broad range of amphipathic organic anions across membranes. MRPs (ABCCs) are one of the highly represented subfamilies of ABC transporters. Plant MRPs also transport various glutathione conjugates across membranes. Arabidopsis thaliana MRP1 is already known to be involved in vacuolar storage of folates. Using heterologously expressed AtMRP1 in yeast and its C-terminal nucleotide binding domain (NBD2) in Escherichia coli, it has been shown that Casein kinase II (CKII) mediated phosphorylation is a potential regulator of AtMRP1 function. AtMRP1 showed enhanced tolerance towards arsenite As(III) in yeast. CKIIII/CKII mediated phosphorylation of AtMRP1 was found to be involved in As(III) mediated signaling. AtMRP1-NBD2 and its serine mutants showed distinct change in secondary structure in the presence of arsenite and methotrexate (MTX) controlled by serine triad phosphorylation. Results showed that AtMRP1 is important for vacuolar accumulation of antifolates as well as tolerance against arsenic, both of which involved phosphorylation in the serine triads at the C terminal NBD of AtMRP1. The experiments provide an important insight into the role of AtMRP1 serine triad phosphorylation under AsIII stress conditions.