Biosynthesis of squalene in the plant chassis has broad application prospects, and identifying efficient enzymes is of great importance. Here, we analyzed the function of squalene synthase genes WsSQS and WsSQS2 from Withania somnifera for squalene biosynthesis in tobacco. WsSQS and WsSQS2 shared 93.7% amino acid (aa) similarity, with divergent residues related to catalysis, NADPH binding, and membrane anchoring. Heterologous expression of WsSQS and WsSQS2 in tobacco increased squalene content by 2.05-fold and 1.68-fold, respectively, with the OE-WsSQS lines reaching 3.19 μg/g DW and the OE-WsSQS2 lines reaching 2.58 μg/g DW, compared to the control plants. Further transcriptomic assays revealed that overexpression of WsSQS induced broader transcriptional changes in the squalene metabolic pathway than WsSQS2. Specifically, the overexpression of WsSQS up-regulated AACT, HMGS, MVD, IspE, FPPS1, FPPS2, and SQS upstream of squalene biosynthesis and down-regulated GGPPS3 downstream of FPP biosynthesis, which is the direct precursor of squalene biosynthesis, while WsSQS2 exerted a more targeted impact, primarily up-regulating HMGS and the key rate-limiting enzyme gene HMGR in the squalene biosynthesis pathway. These findings are consistent with the high efficiency of WsSQS in squalene biosynthesis in tobacco. In summary, this study provides fundamental molecular and biochemical insights into the utilization of heterologous SQSs for squalene production based on the tobacco chassis.