Cardiac catheterizations in horses are mainly performed in the right heart, as access to the left heart traditionally requires an arterial approach. Transseptal puncture (TSP) has been adapted for horses but data on follow-up and closure of the iatrogenic atrial septal defect (iASD) are lacking. To perform TSP and assess postoperative complications and iASD closure over a minimum of 4 weeks. Eleven healthy adult horses. Transseptal puncture was performed under general anesthesia. Serum cardiac troponin I concentrations were measured before and after puncture. Weekly, iASD closure was monitored using transthoracic and intracardiac echocardiography. Relationship between activated clotting time and anti-factor Xa activity during postoperative enoxaparin treatment was assessed in vitro and in vivo. Transseptal puncture was successfully achieved in all horses within a median duration of 22 (range, 10-104) minutes. Balloon dilatation of the puncture site for sheath advancement was needed in 4 horses. Atrial arrhythmias occurred in 9/11 horses, including atrial premature depolarizations (N = 1), atrial tachycardia (N = 5), and fibrillation (N = 3). Serum cardiac troponin I concentrations increased after TSP, but remained under the reference value in 10/11 horses. Median time to iASD closure was 14 (1-35) days. Activated clotting time correlated with anti-factor Xa activity in vitro but not in vivo. Transseptal puncture was successfully performed in all horses. The technique was safe and spontaneous iASD closure occurred in all horses. Clinical application of TSP will allow characterization and treatment of left-sided arrhythmias in horses.