Surfactin, a good biological surfactant, is derived from the metabolites of microorganisms. However, the ability of natural strains to produce surfactin is low, and so the presented study aimed to use a novel mutagenesis technology to increase their yields. Atmospheric and room temperature plasma (ARTP) was used to conduct mutation breeding of Bacillus subtilis CICC 10721, and a mutant strain M45 with a higher surfactin yield of 34.2% and a stable subculture was screened out. From the fermentation kinetics study, it was found that the maximum cell dry weight, maximum growth rate and surfactin synthesis parameters of the mutant strain M45 were all greater than that of the original strain. Scanning electron microscope and laser scanning confocal microscope observations showed that the spore morphology changed after ARTP treating, and the intracellular Ca2+ concentration of the mutant increased. Genome resequencing analysis showed that 66 single nucleotide poymorphism non-synonymous mutation sites occurred in M45, and the identification results of the fermentation broth extract from M45 showed that it is composed of C12 -C16 surfactin. ARTP mutagenesis was found to change the morphology of bacteria, membrane permeability and genes related to the synthesis and secretion of surfactin. The present study provides a basis for industrial production of surfactin and an understanding of the mutagenesis mechanism. © 2021 Society of Chemical Industry.