This study reports the initial in vivo use of a combined radiofrequency ablation and cryo-anchoring (RFC) catheter as an alternative to surgical mitral valve (MV) leaflet resection. Radiofrequency ablation thermally shrinks enlarged collagenous tissues, providing an alternative to leaflet resection, and cryo-anchoring provides reversible attachment of a catheter to freely mobile MV leaflets. Excised porcine MVs (n = 9) were tested in a left heart flow simulator to establish treatment efficacy criteria. Resected leaflet area was quantified by tracking markers on the leaflet surface, and leaflet length reductions were directly measured on echocardiography. Leaflet area decreased by 38 ± 2.7%, and leaflet length decreased by 9.2 ± 1.8% following RFC catheter treatment. The RFC catheter was then tested acutely in healthy pigs (n = 5) under epicardial echocardiographic guidance, open-chest without cardiopulmonary bypass, using mid-ventricular free wall access. Leaflet length was quantified using echocardiography. Quantitative assessment of MV leaflet length revealed that leaflet resection was successful in 4 of 5 pigs, with a leaflet length reduction of 13.3 ± 4.6%. Histological, mechanical, and gross pathological findings also confirmed that RFC catheter treatment was efficacious. The RFC catheter significantly reduces MV leaflet size in an acute animal model, providing a possible percutaneous alternative to surgical leaflet resection.