In order to discover more natural products possessing potentially antiepileptic activities, three C21 steroids, including a new one, characterized as caudatin-3-O-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside (1), and two known analogs, otophylloside B (2) and caudatin-3-O-β-D-oleandropyranosyl-(1 → 4)-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside (3), were isolated from the chloroform extract of the roots of Cynanchum otophyllum and evaluated for their antiepileptic activities by pentylenetrazole (PTZ)-induced zebrafish larval locomotor assay. The results showed that all of them had marked activities of suppressing PTZ-induced seizure behaviors in larval zebrafish at the dose of 10 μg/ml.