Abstarct: Cold stress is one of the important abiotic stresses, which has endangered the production of agricultural products. Understanding the response of plants to cold stress increases human knowledge about the mechanisms of increasing tolerance in plants and the development of resistant cultivars. The aim of this study was to investigate the effect of titanium dioxide nanoparticle on tolerance induction in fenugreek under cold stress and to evaluate the exxpression profile of some genes involved in the biosynthesis pathway of diosgenin. Treatments in this experiment included temperature (23 and 10°C), different harvest times (6, 24 and 48 hours) and different levels of titanium dioxide nanoparticle (0, 2, 5 and 10 ppm). The results of this study showed that cold stress leads to degradation and decomposition of chlorophyll and carotenoid content. In addition, under cold stress, the activity of antioxidant enzymes such as catalase, guaiacol peroxidase and ascorbate peroxidase was increased. Under cold stress, the percentage of electrolyte leakage and malondialdehyde content due to cell membrane vulnerability and oxidation of fatty acids was increased significantly compared to normal temperature as well. The exxpression of SEP, SQS, CAS and SSR genes under low temperature stress and titanium dioxide nanoparticle treatment was up-regulated by 11, 33, 21 and 16 times, respectively, compared to normal temperature (no application of titanium dioxide nanoparticles). In addition, for most physiological traits, concentration of 2 ppm and harvest time of 48 hours had the greatest effect, while in gene exxpression, concentrations of 5 and 10 and harvest times of 24 and 48 stimulated gene exxpressions more effectively. Overall, the results of this study revealed that the application of titanium dioxide could effectively stimulate the biosynthesis of primary and secondary mextabolites and thus induce tolerance to cold stress in fenugreek. In addition, the results of the present study might be applied in the mextabolic engineering of diosgenin in fenugreek.