Although AIMP3/p18 is normally associated with multi-tRNA synthetase complex via specific interaction with methionyl-tRNA synthetase, it also works as a tumor suppressor via the stimulatory interaction with ATM, the upstream kinase of p53 in response to DNA damage. To understand its molecular interaction and working mechanism, we determined the crystal structure of AIMP3 at 2.0 Å resolution and the potential site involved in its interaction with ATM. AIMP3 contains the two distinct domains linked by 7 aa (K57-S63) forming 310 helix. The 56aa N-terminal domain consists of 2 helices to which three antiparallel -strands are inserted and 111 aa C-terminal domain contains a bundle of five helices (T64-Y152) with a following coiled region (P153-L169). Structural analyses unveiled homologous proteins such as yeast glutamyl-tRNA synthetase, Arc1p, EF1B and glutathione S-transferase and suggested two potential molecular interaction sites. Among them, mutations at the C-terminal putative binding site abolished the interaction of AIMP3 with ATM, but not with methionyl-tRNA synthetase . These mutants also have lost their ability to activate p53. Thus, this work suggests that AIMP3 would bind to two different target proteins via different sites and residues critical for its tumor suppressive activity via ATM.