Limosilactobacillus fermentum KUB-D18 is a heterofermentative lactic acid bacterium that its potential probiotic relevance originally isolated from the chicken intestine. This study sequenced a whole-genome of L. fermentum KUB-D18 and annotated its genes and functions in relation to probiotic properties. As a result, the genome sequence of L. fermentum KUB-D18 approximately contained 2.02 Mbps with GC content of51.7%. After annotating the genome by integrated protein and pathway databases, 2,158 protein-encoding genes were majorly annotated for metabolisms of amino acids, carbohydrates and cofactors as well as vitamins which showed a versatile metabolic capability to gastrointestinal microhabitats. According to the comparative genome analysis of L. fermentum KUB-D18 and the other related strains, L. fermentum KUB-D18 showed common characteristics e.g., folate biosynthesis and bile salt hydrolase enzymes-related cholesterol lowering effect as well as a unique gene cluster involved in metabolism of l-ascorbic acid of L. fermentum KUB-D18. Taken together, L. fermentum KUB-D18 genome provides the genetic basis towards cellular capability for further elucidating the functional mechanisms of its probiotic properties. This study serves for designing desirable targets for the development of probiotic foods and feeds.