Neisseria meningitidis, the meningococcus, bears the potential of life-threatening invasive diseases, but it usually colonizes the nasopharynx without causing any symptoms. Within the nasopharynx, Neisseria meningitidis must face temperature changes depending on the ambient air temperature. Indeed, the nasopharyngeal temperature can be substantially lower than 37°C, the temperature commonly used in experimental settings. Here, we compared meningococcal biofilm formation, autoaggregation and cellular adherence between 32°C and 37°C and found a clear increase in all these phenotypes at 32°C, suggestive for a stronger in vivo colonization capability at this temperature. A comparative proteome analysis approach revealed differential protein expression levels between 32°C and 37°C, predominantly affecting the outer membrane. Among 375 analyzed proteins, 75 were localized in the outer membrane, 37 in the periplasm, 48 in the inner membrane and 215 in the cytosol. The outer membrane proteins NHBA, NMB1030 and ACP showed strongest upregulation at 32°C and were partially responsible for the observed temperature-dependent phenotypes. Screening of different global regulators of Neisseria meningitidis revealed that the extracytoplasmic sigma factor, E, might be involved in the temperature-dependent biofilm formation. In conclusion, subtle temperature changes trigger adaptation events promoting mucosal colonization by meningococci