Background: The ideal fraction of nitrous oxide (N2O) in oxygen (O2) for rapid lung collapse remains unclear. Accordingly, this prospective trial aimed to determine the 50% effective concentration (EC50) and 95% effective concentration (EC95) of N2O in O2 for rapid lung collapse. Methods: This study included 38 consecutive patients undergoing video-assisted thoracoscopic surgery (VATS). The lung collapse score (LCS) of each patient during one-lung ventilation was evaluated by the same surgeon. The first patient received 30% N2O in O2, and the subsequent N2O fraction in O2 was determined by the LCS of the previous patient using the Dixon up-and-down method. The testing interval was set at 10%, and the lowest concentration was 10% (10%, 20%, 30%, 40%, or 50%). The EC50 and EC95 of N2O in O2 for rapid lung collapse were analyzed using a probit test.Results: According to the up-and-down method, the N2O fraction in O2 at which all patients exhibited successful lung collapse was 50%. The EC50 and EC95 of N2O in O2 for rapid lung collapse were 27.7% (95% confidence interval 19.9%–35.7%) and 48.7% (95% confidence interval 39.0%–96.3%), respectively.Conclusions: In patients undergoing VATS, the EC50 and EC95 of N2O in O2 for rapid lung collapse were 27.7% and 48.7%, respectively.Trial registration: http://www.chictr.org/cn/ Identifier ChiCTR19 00021474, registered on 22 February 2019