[Background]BacillusLFB112 is a strain ofBacillus amyloliquefaciensscreened in our laboratory. Previous studies found that it has a strong ability for fatty acid metabolism and can improve the lipid metabolism of broilers when used as feed additives. [Methods] This study aimed to confirm the fatty acid metabolism ofBacillusLFB112. Sterilized soybean oil (SSO) was added to the Beef Peptone Yeast (BPY) medium, and its effect on fatty acid content in the supernatant and bacteria, as well as expression levels of genes related to fatty acid metabolism, were studied. The control group was the original culture medium without oil. [Results] Acetic acid produced by the SSO group ofBacillusLFB112 decreased, but the content of unsaturated fatty acids increased. The 1.6% SSO group significantly increased the contents of pyruvate and acetyl-CoA in the pellets. Furthermore, the mRNA levels of enzymes involved in the type II fatty acid synthesis pathway of FabD, FabH, FabG, FabZ, FabI, and FabF were up-regulated. [Conclusions] Soybean oil increased the content of acetyl-CoA inBacillusLFB112, activated its type II fatty acid synthesis pathway, and improved the fatty acid metabolism level ofBacillusLFB112. These intriguing results pave the way for further investigations into the intricate interplay betweenBacillusLFB112 and fatty acid metabolism, with potential applications in animal nutrition and feed additive development.