Abstract: A vasicinol-producing strain of Bacillus methylotrophicus FJAT-16022 was screened from 14 Bacillus-like bacteria with an aid of liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF MS) combined with retrieval comparison against the mass spectrometry database, Metlin. The obtained vasicinol had a matching score of 95.1 with the database, and its relative content was up to 1.73% of the total metabolites in the cultural supernatants. It appeared that B. methylotrophicus FJAT-16022 was a high-yield strain in producing vasicinol. The result obtained paved the way for the development and utilization of vasicinol from the identified bacterium.