As the transseptal (TS) puncture has become an integral part of many types of cardiac interventional procedures, its technique that was initial reported for measurement of left atrial pressure in 1950s, continue to evolve. Our laboratory adopted a modified technique which uses only coronary sinus catheter as the landmark to accomplishing TS punctures under fluoroscopy. The aim of this study is prospectively to evaluate the training and learning process for TS puncture guided by this modified technique. Guided by the training protocol, TS puncture was performed in 120 consecutive patients by three trainees without previous personal experience in TS catheterization and one experienced trainer as a controller. We analysed the following parameters: one puncture success rate, total procedure time, fluoroscopic time, and radiation dose. The learning curve was analysed using curve-fitting methodology. The first attempt at TS crossing was successful in 74 (82%), a second attempt was successful in 11 (12%), and 5 patients failed to puncture the interatrial septal finally. The average starting process time was 4.1 ± 0.8 min, and the estimated mean learning plateau was 1.2 ± 0.2 min. The estimated mean learning rate for process time was 25 ± 3 cases. Important aspects of learning curve can be estimated by fitting inverse curves for TS puncture. The study demonstrated that this technique was a simple, safe, economic, and effective approach for learning of TS puncture. Base on the statistical analysis, approximately 29 TS punctures will be needed for trainee to pass the steepest area of learning curve.