Background— Emerging clinical evidence demonstrates high prevalence of QTc prolongation and complex ventricular arrhythmias in patients with anti-Ro antibody (anti-Ro Ab)–positive autoimmune diseases. We tested the hypothesis that anti-Ro Abs target the HERG (human ether-a-go-go–related gene) K ⁺ channel, which conducts the rapidly activating delayed K ⁺ current, I _Kr , thereby causing delayed repolarization seen as QT interval prolongation on the ECG. Methods and Results— Anti-Ro Ab–positive sera, purified IgG, and affinity-purified anti-52kDa Ro Abs from patients with autoimmune diseases and QTc prolongation were tested on I _Kr using HEK293 cells expressing HERG channel and native cardiac myocytes. Electrophysiological and biochemical data demonstrate that anti-Ro Abs inhibit I _Kr to prolong action potential duration by directly binding to the HERG channel protein. The 52-kDa Ro antigen–immunized guinea pigs showed QTc prolongation on ECG after developing high titers of anti-Ro Abs, which inhibited native I _Kr and cross-reacted with guinea pig ERG channel. Conclusions— The data establish that anti-Ro Abs from patients with autoimmune diseases inhibit I _Kr by cross-reacting with the HERG channel likely at the pore region where homology between anti–52-kDa Ro antigen and HERG channel is present. The animal model of autoimmune-associated QTc prolongation is the first to provide strong evidence for a pathogenic role of anti-Ro Abs in the development of QTc prolongation. It is proposed that adult patients with anti-Ro Abs may benefit from routine ECG screening and that those with QTc prolongation should receive counseling about drugs that may increase the risk for life-threatening arrhythmias.