Autotomy refers to the voluntary detachment of a body part at a pre-determined breakage plane. The sacrifice is typically predator-initiated and the behaviour facilitates escape. The breakage plane (autotomy plane) is not inherently fragile, but rather its structural integrity is weakened by neural activation of effectors that abruptly reduce tensile strength. Dorsal appendages (‘cerata’) of the nudibranch, Melibe leonina, readily detach when pinched by forceps or by a predatory crab’s cheliped. The ceratal autotomy plane includes a nerve ring that innervates cells with granule-filled cytoplasmic processes extending to all connective tissue structures crossing the autotomy plane. Degranulation of these cells may disrupt collagenous connective tissues, including epithelial basal laminae, during autotomy behaviour. In addition, the nerve ring and associated granule-filled cells of the autotomy plane are flanked by a pair of sphincter muscles. When a ceras is pinched, a long-lasting train of synchronized action potentials is initiated within neurons of a ganglion at the base of the ceras, the sphincter muscles contract strongly, granule-filled cells degranulate, and tissues of the autotomy plane separate. The ability to autotomize a ceras emerges at a specific stage of juvenile development – stage III. We therefore hypothesized that the distinctive, granule-filled cells of the autotomy plane would differentiate at stage III. By immunolabeling nerves and phalloidin-labeling muscles, together with transmission electron microscopy, we found that granule-filled cells were present at the prospective autotomy plane well before stage III, but the sphincter muscles differentiated at the onset of stage III. We conclude that the sphincter muscles are an essential morphological component of autotomy behaviour.