Environmental temperature is an essential physical quantity that substantially influences cell physiology by changing the equilibria and kinetics of biochemical reactions occurring in cells. Although it has been extensively used as a readily controllable parameter in genetic and biochemical research, much remains to be explored about the temperature responses of intracellular biomolecules in vivo and at the molecular level. Here we report in vivo probing, achieved with label-free Raman microspectroscopy, of the temperature responses of major intracellular components such as lipids and proteins in living fission yeast cells. The characteristic Raman band at 1602 cm1, which has been attributed mainly to ergosterol, showed a significant decrease (approximate to 47%) in intensity at elevated temperatures above 35 degrees C. In contrast to this high temperature sensitivity of the ergosterol Raman band, the phospholipid and protein Raman bands did not vary much with increasing culture temperature in the 2638 degrees C range. This finding agrees with a previous biochemical study that showed that the initial stages of ergosterol biosynthesis in yeast are hindered by temperature elevation. Moreover, our result demonstrates that Raman microspectroscopy holds promise for elucidation of temperature-dependent cellular activities in living cells, with a high molecular specificity that the commonly used fluorescence microscopy cannot offer.