The optimum growth of soil crust-forming cyanobacterial species occurs between 21 and 30 °C. When the temperature decreases below _5 °C, the liquid water in the cyanobacterial cells may freeze. In the natural environment, the temperature gradually decreases from autumn to winter, and the diurnal temperatures fluctuate enormously. It was hypothesized that the physiology of cyanobacterial cells changes in later autumn to acclimatize the cells to the upcoming freezing temperatures. In the present study, an incubation experiment in growth chambers was designed to stimulate the responses of cyanobacterial cells to diurnal temperature variations before freezing in late autumn. The results showed that “light” cyanobacterial soil crusts are more tolerant to diurnal temperature fluctuations than “dark” cyanobacterial soil crusts. After the first diurnal temperature cycle between 24 and _4 °C, the malondialdehyde (MDA) contents increased and the photosynthetic activity decreased. The superoxide dismutase activity increased, more extracellular polysaccharides (EPS) were secreted and the ratios of the light-harvesting and lightscreening pigments decreased. With increasing numbers of diurnal temperature cycles, the MDA contents and photosynthetic activity gradually returned to their initial levels. Our results suggest that there are at least three pathways by which crust-forming cyanobacteria acclimate to the diurnal temperature cycles in the late autumn in the Hopq Desert, Northwest China. These three pathways include increased secretion of EPS, regulation of the ratios of light-harvesting and lightscreening pigments, and activation of the antioxidant system. The results also indicate that late autumn is a critical period for the protection and restoration of the cyanobacterial soil crusts in the Hopq Desert.