Toward the establishment of suture closure procedures for atrial septal defect or patent foramen ovale under guidance of three-dimensional (3D) echocardiography but without use of cardiopulmonary bypass (off-pump surgery), an experimental study was conducted using a laparoscopic suture instrument, Maniceps. First, the panel setting of the 3D echo system which was optimal for precisely visualizing the surgical instruments on the image display with the least time delay was determined. The optimal setting was: 1) harmonic imaging, 2) no smoothing, 3) low scanning line density, and 4) a scanning range around 55 degrees. Using an ex vivo model of atrial septal defect, 3D echo-guided surgical procedures were attempted in three steps. First, grasping of the edge of the defect with a forceps was attempted. It was feasible in every direction. Reverberation artifact occasionally disturbed imaging of the defect edge. Second, transfixion suture of the facing edges was attempted. Guided by 3D echo, serial sutures were feasible, but interlocking of the thread was a pitfall. Third, continuous suture of the defect was attempted under 3D echo guidance. Following the initial suture bite on one side, continuous suture could be performed under echo guidance. Deformity of the Maniceps needle after repeated sutures was a limitation. In conclusion, suture closure of the defect under 3D echo guidance using the Maniceps system is feasible in an ex vivo ASD model as visualization is optimized by panel setting for guiding surgical procedures.