Ecdysone receptor (EcR) is an important target for pesticide design. Ligand binding regulates EcR transcriptional activity similar to other nuclear receptors; however, the pathways by which ligands enter and leave the EcR remain poorly understood. Here, we performed computational studies to identify unbinding pathways of an ecdysone agonist [the selective ecdysone agonist, BYI06830] from the EcR ligand binding domain (EcR LBD). BYI06830 can dissociate from EcR LBD via four different pathways with little effect on receptor structure. By comparing the potential of mean force (PMF) of four pathways, path 2 was considered to be the most likely exit path for BYI06830, which was located in the cleft formed by the H3-H4 loop, H6-H7 loop, and the H11 C-terminus. Furthermore, structural features along path 2 were analyzed and the structural snapshots of the metastable and transition states were isolated to illustrate the unbinding mechanism of ecdysone agonist from EcR LBD.