The objective of this study was to investigate novel and optimal landmarks for subclavian vein (SV) cannulation. We conducted the study in three phases: (a) Various possible markers for SV cannulation were evaluated by anatomic measurements in patients from intensive care unit and the course of SV was evaluated by radiologic means; (b) Results acquired by the two means were compared and adjusted, then innovative landmarks and a new approach for SV cannulation was proposed; (c) The efficacy of new approach for SV cannulation was compared with that of a traditional one by a prospective, randomized, controlled study. Point A (the junction of the lateral border of sternocleidomastoideus clavicular head and inferior border of clavicle), point B (a point on the lower border of clavicle just above the middle of the line joining the coracoid process and midline of the body), and point D (where SV crosses the inferior border of clavicle) were close in proximity. Points A and B could be considered as innovative landmarks for SV cannulation. Beginners using the new method had significantly higher success rate (86.9% vs. 70.2%, p = 0.008); the time consumed by new method was significant shorter than that by traditional one (5.9 vs. 10.4 min, p = 0.001). Points A and B could be considered as landmarks for identifying puncture site for SV cannulation. Beginners using innovative landmarks for SV cannulation could significantly reduce operation time and increase success rate.