Background: Radiofrequency ablation has been successfully applied to treat right atrial arrhythmias in horses. Ablation of left-sided arrhythmias requires a retrograde transarterial approach which is complicated. In human medicine, the left atrium is accessed through transseptal puncture (TSP) of the fossa ovalis (FO) using a caudal approach via the femoral vein. Objectives: To develop a zero fluoroscopy TSP technique for horses using a jugular vein (cranial) and transhepatic (caudal) approach. Study design: In vivo experimental study. Methods: Transseptal puncture was performed in 18 horses admitted for euthanasia and donated for scientific research under general anaesthesia: using a jugular vein approach (ten horses), a transhepatic approach (two horses) or both (six horses). Radiofrequency energy was applied on a guidewire to perforate the FO and allow sheath advancement under intracardiac and transthoracic echocardiographic guidance. Puncture lesions were inspected post-mortem. Results: Transseptal puncture was successful in 17/18 horses, of which 15/16 jugular vein approaches and 5/8 transhepatic approaches. Failure was due to technical malfunction, inability to advance the guidewire towards the heart and inability to advance the sheath through the FO. Intracardiac echocardiography was essential to safely guide the puncture process. Atrial arrhythmias caused by the TSP occurred in 13/18 horses. Puncture lesions were found in the right atrium in the FO region, and in the left atrium ventral to pulmonary vein ostium III. Main limitations: As in several horses two approaches were tested consecutively, it cannot be excluded that the second TSP was performed at the previous puncture site. Due to the developmental nature of the study the approaches were not randomized and did not allow comparison. Conclusion: Transseptal puncture is feasible in horses using ultrasound guidance and allows for electrophysiological exploration of the left heart. Further studies are needed to evaluate post-operative follow-up.