The histone-like nucleoid structuring (H-NS) protein is conserved in Gram-negative bacteria and plays important roles as a multifunctional bacterial modulator. However, its roles in deep-sea microorganisms warrant investigation. In this study, the relationship between temperature and the regulatory function of H-NS was characterised in the benthic bacterium Shewanella piezotolerans WP3. Comparative microarray analysis of the hns knockout mutant and wild-type strain identified a total of 264 differentially expressed genes at 4°C, which is significantly less than that at 20°C. Notably, H-NS was proved to be involved in the cold-mediated induction of lateral flagellar gene transcription. We further showed that the binding affinity of H-NS is higher at 4°C than 20°C, thus explaining the significant difference in the expression pattern at different temperatures.