The temperature-sensitive gating of human Connexin 26 (hCx26) was analyzed with confocal Raman microscopy. High-resolution Raman spectra covering the spectral range between 400 and 1500 rel. cm−1 with a spectral resolution of 1 cm−1 were fully annotated, revealing notable differences between the spectrum recorded from solubilized hCx26 in Ca2+-buffered POPC at 10°C and any other set of protein conditions (temperature, Ca2+ presence, POPC presence). Spectral components originating from specific amino acids show that the TM1/EL1 parahelix and probably the TM4 trans-membrane helix and the plug domain are involved in the gating process responsible for fully closing the hemichannel.