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The foodborne pathogen Listeria monocytogenes resists environmental stresses by forming biofilms. Because this pathogen transmits between the environment and the host, it must adapt to temperature as an environmental stress. In this study, we aimed to identify which proteins were present depending on the temperature in the biofilms of L. monocytogenes EGD-e. Proteins in the supernatants of biofilms formed at 25°C and 37°C were compared using two-dimensional gel electrophoresis and liquid chromatography with tandem mass spectrometry. The larger number of extracytoplasmic proteins associated with cell wall/membrane/envelop biogenesis was identified from the supernatant of biofilms formed at 25°C (7) than those at 37°C (0). Among the 16 extracytoplasmic proteins detected only at 25°C, three were peptidases, namely Spl, Cwh, and Lmo0186. Moreover, mRNA expression of the three peptidases was higher at 25°C than at 37°C. Interestingly, this adaptation of gene expression to temperature was present in sessile cells but not in dispersed cells. After inhibiting the activity of extracytoplasmic peptidases with a protease inhibitor, we noted that the levels of biofilm biomass increased with higher concentrations of the protease inhibitor only when L. monocytogenes grew biofilms at 25°C and not at 37°C. Overall, our data suggest an effect of temperature on the presence of peptidases in L. monocytogenes biofilms. Additionally, increasing the levels of extracytoplasmic peptidases in biofilms is likely a unique feature for sessile L. monocytogenes that causes a naturally occurring breakdown of biofilms and facilitates the pathogen exiting biofilms and disseminating into the environment.