A recessive mutation hsi2 of Arabidopsis (Arabidopsis thaliana) expressing luciferase (LUC) under control of a short promoter derived from a sweet potato (Ipomoea batatas) sporamin gene (Spo(min)LUC) caused enhanced LUC expression under both low- and high-sugar conditions, which was not due to increased level of abscisic acid. The hsi2 mutant contained a nonsense mutation in a gene encoding a protein with B3 DNA-binding domain. HSI2 and two other Arabidopsis proteins appear to constitute a novel subfamily of B3 domain proteins distinct from ABI3, FUS3, and LEC2, which are transcription activators involved in seed development. The C-terminal part of HSI2 subfamily proteins contained a sequence similar to the ERF-associated amphiphilic repression (EAR) motif. Deletion of the C-terminal portion of HSI2 lost in the hsi2 mutant caused reduced nuclear targeting of HSI2. Null allele of HSI2 showed even higher Spo(min)LUC expression than the hsi2 mutant, whereas overexpression of HSI2 reduced the LUC expression. Transient coexpression of 35SHSI2 with Spo(min)LUC in protoplasts repressed the expression of LUC activity, and deletion or mutation of the EAR motif significantly reduced the repression activity of HSI2. These results indicate that HSI2 and related proteins are B3 domain-EAR motif active transcription repressors.