Objective To explore a method for transseptal puncture guided by intracardiac echocardiography (ICE) in canines. Methods Transseptal puncture was performed in 10 healthy canines weighing 10-15 kg guided by ICE. Anesthesia was induced by intramuscular injection of atropine and propofol. Isoflurane was used to maintain anesthesia after trachea cannula. After percutaneous puncture of the bilateral femoral veins, a sheath was inserted. Then, an transseptal puncture needle was delivered to the superior vena cava via the right sheath and pointing to the 12 o'clock direction. An ICE catheter was delivered to the right atrium via the left sheath to construct a "HomeView". A P-bend was made in the ICE catheter, and the catheter was rotated clockwise to reveal the aortic root, atrial septum, and fossa ovalis. The ICE catheter was adjusted so that the fossa ovalis was located in the center of the section before the catheter was secured. The transseptal puncture needle and sheath were withdrawn and slowly rotated clockwise to the 3 o'clock position. The "tent sign" was visible once the needle and sheath reached the fossa ovalis. Once the sheath was fixed, the needle was inserted. The "tent sign" disappeared after crossing the atrial septum. An intravenous bolus of heparin saline was administered to confirm that the needle had reached the left atrium, fixing the needle and pushing the sheath, and the "double track sign" was visible. After withdrawing the needle and flushing the tube with heparin saline, two and a half coils of guidewire were placed in the left atrium, which were visible in the section. After surgery, 5 canines were sacrificed and observed for the presence of pericardial cavity effusion and the puncture site, and the other canines were followed up for 30 days and the complications were recorded. Results All transeptal punctures were successful in 10 canines, including single puncture in 9 canines. The procedure duration was (5.2±1.4) min, and the exposure duration was (24.9±4.5) s. Five canines were sacrificed immediately after the operation. No bloody pericardial effusion was found and the sites of transseptal puncture were at the center of fossa ovalis. There were no complications in the other canines 30 d after operation. Conclusion Transseptal puncture guided by ICE in canines is easily performed, with high efficacy and safety.