Notch signaling is central to cell differentiation, organ development, and apoptosis. Upon ligand binding, the Notch intracellular domain (NotchIC) translocates to the nucleus to interact with its DNA-binding partner, RBP-Jkappa. The NotchIC-RBP-Jkappa complex activates target genes, such as those encoding the Hrt and Hes families of basic-helix-loop-helix (bHLH) transcriptional repressors. Hrt transcripts are enriched in the developing cardiovascular system, and mice lacking Hrt2 have cardiac malformations. Here we show that Hrt2 and Hes1 interact with RBP-Jkappa to negatively regulate Notch-dependent activation of Hrt and Hes expression. The bHLH domain of Hrt2 was necessary for this interaction, and disrupting the protein complex abrogated the negative autoregulation. The interaction did not interfere with the formation or DNA-binding of the NotchIC-RBP-Jkappa complex, indicating direct inhibition by Hrt and Hes as co-repressors. These findings suggest a novel mechanism for negative feedback on Notch signaling that requires RBP-Jkappa to interact physically with Hrt and Hes.