AbstractThe human ERG protein (HERG or Kv11.1) encoded by the human ether‐a‐go‐go‐related gene (herg) is the pore‐forming subunit of the cardiac delayed rectifier potassium current (IKr) responsible for action potential (AP) repolarization. Mutations in HERG lead to long‐QT syndrome, a major cause of arrhythmias. Protein–protein interactions are fundamental for ion channel trafficking, membrane localization, and functional modulation. To identify proteins involved in the regulation of the HERG channel, we conducted a yeast two‐hybrid screen of a human heart cDNA library using the C‐terminus or N‐terminus of HERG as bait. Fifteen proteins were identified as HERG amino terminal (HERG‐NT)‐interacting proteins, including Caveolin‐1 (a membrane scaffold protein with multiple interacting partners, including G‐proteins, kinases and NOS), the zinc finger protein, FHL2 and PTPN12 (a non‐receptor tyrosine phosphatase). Eight HERG carboxylic terminal (HERG‐CT)‐interacting proteins were also identified, including the NF‐κB‐interacting protein myotrophin, We have identified multiple potential interacting proteins that may regulate cardiac IKr through cytoskeletal interactions, G‐protein modulation, phosphorylation and downstream second messenger and transcription cascades. These findings provide further insight into dynamic modulation of HERG under physiological conditions and arrhythmogenesis.