Abstract Background The most effective approach for long-standing persistent atrial fibrillation (LPAF) ablation remained undetermined. Our goal was to explore the heterogeneous left atrial substrate in patients with LPAF and to evaluate the effectiveness of a novel individualized substrate modification (ISM) approach in LPAF ablation. Methods One hundred and twenty-four patients with LPAF were randomized to ISM group (n=64) or stepwise ablation (SA) group (n=60). After pulmonary vein isolation, ISM was performed in the ISM group and SA was applied in the SA group. The clinical effectiveness after a single and a repeated procedure was compared. Results The total procedural time was significantly shorter in ISM than that in SA. In the ISM group, mild left atrial substrate was observed in 17 (27.4%), moderate in 26 (41.9%) and severe in 19 (30.6%) patients after successful cardioversion of the 62 patients. The intention-to-treat analysis showed that sinus rhythm was maintained in 65.5% of patients in the ISM group and in 45.0% of patients in the SA group after a single procedure, P=0.04. Atrial tachycardia (AT) recurred in 5 of 22 in the ISM group and in 20 of 33 in the SA group, P=0.01. After a repeated procedure, 75% of patients in the ISM group and 63.3% of patients in the SA group were free of further recurrence, P=0.16. Conclusions Left atrial substrate varied noticeably in patients with LPAF. The ISM approach was superior to SA approach in terms of procedural time, recurrence rate of AT and clinical effectiveness after a single procedure. However, they yielded comparable outcomes after a repeated procedure.