Histone-like protein HU is essential for DNA recombination, repair, and transcriptional regulation in bacteria. However, the physiological roles of HU proteins in Listeria monocytogenes (LmHU) remain unexplored. Given the significant biofilm-forming ability of this foodborne pathogen and its associated cross-contamination risks, identifying novel control targets is critical. LmHU, as the sole double-stranded DNA-binding protein in L. monocytogenes, is a promising candidate. This study systematically explored its contributions to biofilm formation, motility, and the regulation of virulence factors. The results indicated that high levels of LmHU in vivo promoted cell cohesion, leading to a chain-like structure among L. monocytogenes. Additionally, LmHU could be secreted into the biofilm matrix, reinforcing the structure by interacting with extracellular polymeric substances. However, elevated LmHU levels inhibited bacterial motility, flagellar synthesis, and host invasion in Caco-2 cells. RNA-seq analysis revealed 374 differentially expressed genes in the Lmhu mutant relative to the wild-type strain, supporting these findings. Further enrichment analysis and validation experiments suggested that Lmhu overexpression impaired trehalose utilization. These results indicate that LmHU may serve as a potential target for novel disinfectants or therapies to reduce risks associated with L. monocytogenes in food safety and public health.