A new lycosinine derivative, 9-O-demethyllycosinine B, was isolated from the native Brazilian Hippeastrum breviflorum Herb., Amaryllidaceae, along with the well-known alkaloids lycosinine B and lycorine. The structure of the new compound was established by physical and spectroscopic methods. 9-O-demethyllycosinine B is the third lycosinine variant identified in the Amaryllidaceae family.