Background Fever is associated with the manifestation of Brugada phenotype and ventricular tachycardia/ventricular fibrillation (VT/VF) in patients with Brugada syndrome (BrS). The thermal effect on the pathogenesis of functional substrates in BrS remains unknown. Objective This study aimed to elucidate the thermal effect on BrS phenotype, VT/VF, and electrophysiological characteristics of epicardial functional substrates in BrS. Methods We consecutively studied 15 patients with BrS receiving radiofrequency catheter ablation for drug-refractory ventricular tachyarrhythmias. Baseline characteristics, electrocardiographic features, and changes in epicardial functional substrates before and after epicardial warm water instillation (n = 6) were recorded and analyzed. Results A total of 15 male patients (mean age 41.3 ± 10.3 years) with type 1 BrS presenting with ventricular tachyarrhythmias were consecutively enrolled. Epicardial mapping in 11 patients demonstrated a significantly larger epicardial scar/low-voltage zone (LVZ) area within the right ventricular outflow tract and anterior right ventricular free wall than within the endocardium (6.32 ± 12.74 cm2 vs 52.91 ± 45.25 cm2; P =.007). Epicardial warm water instillation in 6 patients led to a significant enlargement of the functional scar/LVZ area (123.83 ± 35.26 cm2 vs 63.53 ± 40.57 cm2; P =.03), accelerated conduction velocity of the endocardium and epicardium without scar/LVZ area, and increased VT/VF inducibility (16.7% vs 100%; P =.02). Ablation by targeting premature ventricular complexes and/or epicardial abnormal substrates rendered noninducibility of VT/VF and prevented the recurrences of VT/VF. Conclusion Epicardial warm water instillation enhanced functional epicardial substrates, which contributed to the increased inducibility of ventricular tachyarrhythmias in BrS. Ablation by targeting the triggers and abnormal epicardial substrates provided an effective strategy for preventing ventricular tachyarrhythmia recurrences in BrS.