There are currently no prospective, controlled trials of needle puncture speed in EUS-guided FNA (EUS-FNA). In this study, we prospectively evaluated the accuracy of histological diagnosis and the tissue acquisition rate of EUS-FNA by using the door-knocking method (DKM) with a standard 22-gauge needle.From November 2013 to August 2014, 82 patients who had solid pancreatic masses underwent EUS-FNA in which the conventional method (CM) and DKM with 2 respective passes in turn were used. The primary outcomes of this study were the accuracy of histological diagnosis and the rates of tissue acquisition in 2 FNA procedures by using these 2 methods.Although the successful tissue acquisition rate for histology was not significantly different with the DKM and CM (91.5% vs 89.0%, P = .37), the high cellularity tissue acquisition rate for histology with the DKM was significantly superior to that with the CM (54.9% vs 41.5%, P = .03). However, adequate quality rate and accuracy were not different in the DKM and CM (78.0% vs 80.5%, P = .42 and 76.8% vs 78.0%, P = .50, respectively). In the transgastric puncture group, although the adequate quality rate and accuracy were similar in the DKM and CM (84.1% vs 79.4%, P = .30 and 84.1% vs 76.2%, P = .11, respectively), the tissue acquisition rate tended to be higher with the DKM than the CM (93.7% vs 85.7%, P = .06). Moreover, the high cellularity tissue acquisition rate was significantly better with the DKM than the CM (63.5% vs 39.7%, P = .002). On the other hand, in the transduodenal puncture group, although the tissue acquisition rate was similar with the DKM and CM (84.2% vs 100%, P = .13), the adequate quality rate and accuracy were significantly lower with the DKM than with the CM (57.9% vs 84.2%, P = .03 and 52.6% vs 84.2%, P = .02, respectively).EUS-FNA by using a 22-gauge needle with the DKM did not improve the accuracy of histological diagnosis, but enabled acquisition of a larger amount of tissue specimen by using transgastric puncture. (http://www.umin.ac.jp/english/: UMIN000012127.).