Abstract： Background Previous studies have investigated the technique of linear ablation at the mitral isthmus (MI) in patients with idopathic atrial fibrillation (AF), but MI ablation in patients with prosthetic natural mitral valves (MVs) was not described in detail. Present study sought to summarize our initial experience of ablating MI in patients with prosthetic MVs Methods Patients with drug refractory AF and prosthetic MVs were eligible for this study, and the patients with natural MVs but received MI ablation served as control group. Left atrium (LA) mapping and ablation was carried out guided by CARTO system. The anatomy of MI was assessed via computer topography scan.Results During the study period, a consecutive of 19 patients (male/female=12/7, mean age of (48±-6) years) with prosthetic MVs (16 with metal valves, 3 with biologic valves) entered for AF ablation, other 35 patients served as control group. In study group, mapping along MI documented lower voltages ((2.0±1.0) vs. (3.1±1.3) mV, P=0.002), more fragmented potentials (19/19 vs. 20/15, P<0.001 ), and higher impedance ((132±34) vs. (110±20) Ω, P=0.004). After initial ablation, more residual gaps along the MI lesions were found in study group (2.4±0.4 vs. 1.7±0.3, P <0.001). The mean length of MI ((6.2±3.3) vs. (7.1±2.3) cm, P=0.25) was comparable between 2 groups, but the MI in study group was much thicker ((3.1 ±1.8) vs. (2.1±1.07) cm, P=0.01 ) and all were found as pouch type (19/19 vs. 2/35, P <0.001). The follow-up results were comparable (65.1% vs. 72.3%, P=0.30).Conclusion For patients with prosthetic MVs, linear ablation at MI could be successfully carried out despite anatomical and pathological changes.