Abstract: Lactiplantibacillus pentosus is identified as a promising species capable of fermenting lignocellulose hydrolysate to produce lactic acid, with additional probiotic potential. To fully reveal the genome structure, biological characteristics, evolutionary relationships, and probiotic functions of Lactobacillus pentosus, and provide a scientific basis for the efficient utilization of biomass resources, in this study, the Illumina NovaSeq and PacBio Revio sequencing technologies were employed, combined with bioinformatics software, to perform a comprehensive genome analysis of the strain L. pentosus Lp-16. The analysis included genome assembly, optimization, functional annotation, and prediction. Gene collinearity analysis and the construction of a molecular phylogenetic tree were conducted to explore the biological characteristics and evolutionary relationships of L. pentosus Lp-16, providing a theoretical foundation for lactic acid fermentation and biomass resource utilization. The whole genome analysis showed that L. pentosus Lp-16 was highly enriched in carbohydrate metabolism and amino acid utilization, the COG functional genes contained the largest number of carbohydrate transport and metabolism genes（271）, and the KEGG functional annotations contained the largest number of metabolic classes（927）, indicating that L. pentosus Lp-16 had strong environmental adaptation and metabolic abilities. KEGG enrichment analysis showed that L. pentosus Lp-16 expansion gene family was significantly abundant in carbohydrate metabolic pathways, such as galactose, starch and sucrose metabolism. According to GO database statistics, 13573 genes of L. pentosus Lp-16 have been classified and functional annotations, which have the ability of nitrogen recycling and strong nitrogen metabolism to maintain cell physiological functions. The detection of 57 glucoside hydrolases in the CAZy database indicates that L. pentosus Lp-16 has a strong capacity for carbohydrate metabolism. In this study, we revealed the whole genome information of L. pentosus Lp-16 and preliminarily clarified its specific roles in bacterial survival, host intestinal colonization, and antibacterial activity. These findings lay a theoretical foundation for the development of candidate probiotic strains.