A vacuolar type beta-D-fructofuranosidase (Osbetafruct3) was cloned from etiolated rice seedlings cDNA library. It encodes an open reading frame of 688 residues. The deduced amino acid sequence had 58% identity to the vacuolar type beta-D-fructofuranosidase of maize (Ivr1). Osbetafruct3 exists as a single copy per genome. Northern analyses showed that Osbetafruct3 undergoes organ-specific expression and is involved in the adjustment of plant responses to environmental signals and metabolizable sugars. Osbetafruct3 was also heterologously expressed in Pichia pastoris. The recombinant proteins were confirmed to be a vacuolar type beta-D-fructofuranosidase.