AimsThis study aimed to investigate the potential of Bacillus methylotrophicus as a probiotic. Methods and ResultsA Bacillus isolate designated strain C14 was isolated from Korean traditional fermented soybean paste (doenjang). The strain was identified, and its physiological and biochemical properties were characterized. The gastrointestinal tolerance and immunomodulatory function of strain C14 were also investigated. Strain C14 was identified as B.methylotrophicus by analysis of its biochemical properties using the API 50CHB system and by phylogenetic analysis of the 16S rDNA sequence. Strain C14 showed >80% and >75% of survival for artificial gastric juices (pH 25 and 1% pepsin) and 05% (w/v) bile salt, respectively. Heat-killed B.methylotrophicus C14 inhibited the adhesion of various pathogens and enhanced the adhesion of probiotic bacteria to Caco-2 cells. The heat-killed cells also induced high levels of immune cell proliferation compared with the control and stimulated interleukin-6 and tumour necrosis factor- production in mouse macrophages. ConclusionsBacillus methylotrophicus C14 could be used as a probiotic. Significance and Impact of the StudyRecently identified B.methylotrophicus is a new potential probiotic with high gastrointestinal tolerance.