Dioscorea zingiberensisis a perennial herb famous for the production of diosgenin, which is a valuable initial material for the industrial synthesis of steroid drugs. Sterol C26-hydroxylases, such asTfCYP72A616 andPpCYP72A613, play an important role in the diosgenin biosynthesis pathway. In the present study, a novel gene,DzCYP72A12-4, was identified as C26-hydroxylase and was found to be involved in diosgenin biosynthesis, for the first time inD. zingiberensis, using comprehensive methods. Then, the diosgenin heterogenous biosynthesis pathway starting from cholesterol was created in stable transgenic tobacco (Nicotiana tabacumL.) harboringDzCYP90B71(QPZ88854),DzCYP90G6(QPZ88855) andDzCYP72A12-4. Meanwhile, diosgenin was detected in the transgenic tobacco using an ultra-performance liquid chromatography system (Vanquish UPLC 689, Thermo Fisher Scientific, Bremen, Germany) tandem MS (Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer, Thermo Fisher Scientific, Bremen, Germany). Further RT-qPCR analysis showed thatDzCYP72A12-4was highly expressed in both rhizomes and leaves and was upregulated under 15% polyethylene glycol (PEG) treatment, indicating thatDzCYP72A12-4may be related to drought resistance. In addition, the germination rate of the diosgenin-producing tobacco seeds was higher than that of the negative controls under 15% PEG pressure. In addition, the concentration of malonaldehyde (MDA) was lower in the diosgenin-producing tobacco seedlings than those of the control, indicating higher drought adaptability. The results of this study provide valuable information for further research on diosgenin biosynthesis inD. zingiberensisand its functions related to drought adaptability.