Background <p>Mitral isthmus (MI) line ablation is associated with a higher success rate of ablation for atrial fibrillation (AF), but completely blocking the MI is often challenging. The purpose of this study was to assess the effectiveness and safety of a systematic, step-by-step approach for completely blocking the MI in patients with AF undergoing MI line ablation for the first time.</p> Methods <p>A total of 338 consecutive AF patients who underwent MI ablation for the first time were included in the study. MI line ablation was performed in the following sequence: Step 1 involved endocardial linear ablation from the mitral annulus to the left inferior pulmonary vein; Step 2 involved epicardial ablation in the coronary sinus (CS), next to the endocardial ablation line; Step 3 involved epicardial–endocardial insertion site mapping and ablation; Step 4 involved ethanol infusion of the vein of Marshall (EIVOM); and Step 5 involved ablation of the ostium of the VOM, followed by Step 3 if needed. The complete MI block was evaluated using differential pacing maneuvres.</p> Results <p>After endocardial linear ablation, 111 patients (32.8%) experienced MI block. Further epicardial ablation in the CS induced MI block in another 89 patients (26.3%). In 42 patients (12.4%), epicardial–endocardial insertion sites were ablated to block the MI. EIVOM was performed in 64 patients (18.9%) to achieve MI block. Ablation at the ostium of the VOM induced MI block in 3 patients, and repeating step 3 induced MI block in 17 patients. The total success rate of MI block was 96.4%. No cardiac tamponade occurred during MI ablation.</p> Conclusions <p>With a systematic stepwise approach, acute bidirectional MI block can be achieved with a high success rate and without severe complications.</p> Graphical Abstract <p></p>