Lactobacillus plantarum EM is a probiotic strain with antimicrobial activity, cholesterol-lowering effects, and tolerance to acid and bile. To understand the genetic basis of the probiotic characteristics of this strain, genome sequencing and probiotic-related genetic analysis were performed. The genomic characteristics of L. plantarum EM were confirmed by comparative genomic analysis with 41 probiotic lactic acid bacteria, including 10 L. plantarum strains. L. plantarum EM was shown to contain a circular chromosome of 3,184,808 bp and eight plasmids with various lengths from 5,027 to 76,369 bp. The L. plantarum EM genome had a total of 3560 protein-coding genes, including probiotic-related genes, such as tolerance to acid and bile, temperature stress, and oxidative stress. Comparative genomic analysis showed that L. plantarum EM contained plantaricin and bovicin gene clusters, which are related to antimicrobial activity, and five bile salt hydrolase genes related to serum cholesterol-lowering effects. The genomic analysis confirmed the probiotic properties of L. plantarum EM, and our results indicated that this strain has potential application for use as an industrially important probiotic.