Squalene synthase (SQS) is one of the key enzymes in the synthesis of phytosterols and terpenoids. In order to reveal the function of SQS gene in Dioscorea nipponica, we used the cDNA of D. nipponica as template to clone the DnSQS gene by PCR, and analyzed the obtained sequence by bioinformatics. Simultaneously, the content of diosgenin in different tissues was detected by HPLC, and the expression characteristics of DnSQS gene in different tissues, hormone induction and abiotic stress were analyzed by real-time fluorescence quantitative PCR. The results showed that: (1) two genes (DnSQS1 and DnSQS2) of D. nipponica were successfully cloned. They encode 409 and 433 amino acids, respectively. However, the secondary structures of the encoded proteins consist mostly of α-helix and random coil, both of which have a conserved catalytic core generated by α-helix folding, two functional structures rich in aspartic acid, two transmembrane helix structures and are localized in the endoplasmic reticulum. (2) DnSQS1 and DnSQS2 were closely related to DzSQS, and the evolution of the two DnSQS proteins was reasonably conserved. (3) The contents of diosgenin in different tissues were different, and the levels of the content were in the order of leaf > rhizome > aboveground stem > root; the expression levels of DnSQS1 and DnSQS2 genes in different tissues were also significantly different, with DnSQS1 being expressed at the highest level in aboveground stems and DnSQS2 being expressed at the highest level in leaves. (4) The expression of two DnSQS genes could be induced by MeJA, ABA, SA, Eth, H₂O₂, NaCl and PEG-6000, while GA₃ treatment inhibited the expression of two DnSQS genes. Studies have shown that the two DnSQS genes may be involved in the synthesis of diosgenin and play an important role in the mechanism of adversity stress.