Kluyveromyces marxianus is an aerobic yeast and is interested to be applied in many industries. This research was aimed to study the effect of the sterol alternative to ergosterol on the freezing stress of K. marxianus UBU1-11, a thermotolerant yeast. The 0-9 mgL-1 stigmasterol were added to the YM broth and applied for culturing. The growth of all conditions were not interfered by the addition of stigmasterol. The intra-cellular sterol content was detected in the medium with 5 mgL-1 stigmasterol and higher, where the maximum content was 0.32 mg g-1 cell dry weight. After frozen and thawed, the cultures contained stigmasterol had significantly higher viability than those without. It was found that the amount of stigmasterol contained in cells did not affect the number of survival. The stigmasterol provided a significant protection to the yeast cell when subjected to slow freezing. It also increased the survival rate of the culture subjected to subzero temperature storage.