Objective Via subclavian/jugular vein, successful puncture of interventricular septum (IVS) has been achieved transvenously. However, the approach was limited by acute entry-angle. The study was conducted to investigate a novel transcatheter puncture of IVS via femoral access and transfemoral-venous access to left ventricle (LV) through IVS. Methods Via femoral artery, transcatheter puncture of mid-IVS was performed with a custom-made nickel-titanium needle and 6F-sheath in 16 healthy mini-swine. Then femoral arterio-venous circuit was established through IVS. After pre-dilation of IVS, a 20F-sheath was introduced into LV transvenously over-the-guidewire in 15 swine. Furthermore, transfemoral-venous TAVR was attempted with the approach in another swine. IVS was evaluated postoperatively and was further confirmed pathologically 2 months later. Results All transcatheter puncture of IVS was performed successfully in LV and the mid-IVS thickness was 7.67 +/- 0.98 mm. In all swine, femoral arterio-venous circuit was established via IVS, and a 20F-sheath was introduced into LV and aorta transfemoral-venously (entry-angle: 145.3 +/- 12.2 degrees in front view). After the procedure, there was one swine with moderate tricuspid-regurgitation and five swine with mild residual-shunt (2.6 +/- 0.7 mm). Two months later, residual-shunt was still detected in three swine and the communication was confirmed pathologically. In other swine, no defect occurred and replacement-scar was identified along puncture-tract. In the swine underwent transfemoral-venous TAVR, prosthetic valve was deployed successfully with good function. Conclusions Transfemoral transcatheter puncture of IVS is feasible and safe in a swine model, and large sheath can be introduced into LV transfemoral-venously using the novel access with the aid of vessel circuit.