Background and aim: Trendelenburg positioning (TP) is a common approach used during internal jugular vein (IJV) cannulation. No evidence indicates that TP significantly increases the cross-sectional area (CSA) of the IJV or decreases the overlap between the carotid artery (CA) and the IJV in dialysis patients. The primary aim of this study was to investigate the effects of the TP on the CSA of the right IJV and on its relationship to the CA. Methods: Thirty-seven consecutive hemodialysis patients older than 18 years of age were enrolled. We measured the CSA of the right IJV and overlap rate (at end-expiration at the level of the cricoid cartilage) between the CA and the IJV in two positions: State 0, table flat (no tilt), with the patient in the supine position; State T, in which the operating table was tilted to 15° of TP. Results: Data were collected for all of the 37 patients enrolled in the study. The change in CSA and overlap between the CA and the IJV from the supine to the TP was not significantly different. The CSA was paradoxically decreased in 11 of 37 patients when changed from State 0 to State T. Conclusions: TP does not significantly increase the CSA of the right IJV or decrease the overlap between the CA and the IJV in dialysis patients. In fact, in some patients, it reduces the CSA. Therefore, the use of the TP for IJV cannulation in dialysis patients can no longer be supported.