The DNA-binding protein recombination signal-binding protein-Jk (RBP-J) plays a key role in transcriptional regulation by targeting the intracellular domain of Notch (NIC) and the Epstein-Barr virus nuclear antigen 2 (EBNA2) to specific promoters. In the absence of the Notch signaling, RBP-J acts as a transcriptional suppressor through recruiting co-suppressors such as histone deacetylase (HDAC). KyoT2 is a LIM domain protein that suppresses the RBP-J-mediated transcriptional activation. In the current study, we show that the polycomb group (PcG) protein HPC2, which functions as a transcriptional suppressor, is a candidate of KyoT2-binding proteins. To confirm the physical and functional interaction between KyoT2 and HPC2, we carried out yeast two-hybrid, GST-pull down, co-immunoprecipitation, as well as mammalian two-hybrid assays. Our results showed HPC2 and KyoT2 interacted both in vitro and in vivo, probably through the C-terminal fragment of HPC2 and LIM domains of KyoT2. In addition, we also found that overexpression of HPC2, not only inhibited transactivation of a RBP-J-dependent promoter by NIC, but also transactivation by RBP-J-VP16, a constitutively active form of RBP-J. Taken together, our results suggested that KyoT2 might inhibit the RBP-J-mediated transactivation through NIC by recruiting co-suppressors such as HPC2.