Short infrared laser pulses induce a variety of effects in cells and tissues, including neural stimulation and inhibition. However, the mechanism behind these physiological effects is poorly understood. It is known that the fast thermal gradient induced by the infrared light is necessary for these biological effects. Therefore, this study tests the hypothesis that the fast thermal gradient induced in a cell by infrared light exposure causes a change in the membrane fluidity. To test this hypothesis, we used the membrane fluidity dye, di-4-ANEPPDHQ, to investigate membrane fluidity changes following infrared light exposure. Di-4-ANEPPDHQ fluorescence was imaged on a wide-field fluorescence imaging system with dual channel emission detection. The dual channel imaging allowed imaging of emitted fluorescence at wavelengths longer and shorter than 647 nm for ratiometric assessment and computation of a membrane generalized polarization (GP) value. Results in CHO cells show increased membrane fluidity with infrared light pulse exposure and this increased fluidity scales with infrared irradiance. Full recovery of pre-infrared exposure membrane fluidity was observed. Altogether, these results demonstrate that infrared light induces a thermal gradient in cells that changes membrane fluidity.