The gut microbiota of soymilk intervention is beneficial to maintaining human health. Bacillus subtilis fermented soymilk has brought much interest, due to its richness of thrombolytic nattokinase and the strain of potential probiotic properties. In this study, soymilk was fermented by B. subtilis BSNK-5, and the BSNK-5-fermented soymilk (SMF) on the production of short chain fatty acids (SCFAs) and the regulation of fecal microbiota was initially evaluated by in vitro fecal fermentation. SMF supplementation obviously increased the levels of SCFAs from 32.23 mM to 49.10 mM, especially acetic acid, propionic acid, and isobutyric acid. Additionally, SMF changed the composition and microbial diversity of gut microbiota. After 24 h of anaerobic incubation in vitro, SMF decreased the Firmicutes/Bacteroidota ratio favoring weight loss, increased Lachnospiraceae_UCG-004 and the other beneficial bacteria producing SCFAs, as well as suppressing pathogenic Streptococcus genus. These results revealed the potential use of BSNK-5-fermented soymilk as a potential candidate to promote gut health.