Autotaxin (ATX) is a secreted lysophospholipase D that produces lysophosphatidic acid, a lipid mediator that activates G protein-coupled receptors to evoke various cellular responses. The nuclease-like domain of ATX and the Asn524-linked glycan are reportedly critical for the catalytic activity. Recently, the crystal structures of ATX were determined, but the means by which the nuclease-like domain and the N-glycosylation participate in the catalytic activity still remain undetermined. To address this question, we conducted molecular dynamics simulations of ATX. The simulation trajectories starting from the full-length structure and from structures lacking the nuclease-like domain and/or the glycan were compared. The results suggested that an allosteric interaction pathway, formed by the catalytic domain, including the two insertion regions, the essential glycan modification, and the nuclease-like domain, may stabilize the proper location of the catalytic threonine residue. The results complement those from previous biochemical experiments.