Two new holostan-type glycosides, holotoxin D1 (1) and 25,26-dihydroxy-holotoxin A1 (2), together with two known analogues, stichlorosides C1 (3) and bivittoside D (4), were isolated from the sea cucumber Apostichopus japonicus Selenka. The structures of the new compounds were elucidated on the basis of detailed spectroscopic analysis. Glycosides 1 and 3 exhibited potent antifungal activity.