Mouse Leydig cells(TM3 cells) were treated with Dendrobium officinale extract at mass concentration of 20, 40, 60, 80 and 100 μg/mL for 24 h. The cell proliferation activity was detected by MTS method. The mRNA expression levels of proliferation and apoptosis-related genes, testosterone synthase genes, as well as transcription factor genes and upstream regulatory factor genes relevant to testosterone synthase were detected by qRT-PCR in TM3 cells. The expression of apoptosis-related proteins and testosterone synthases was analyzed by Western blot. And the testosterone secretion levels were detected by ELISA. The results showed that treatment with 40 μg/mL Dendrobium officinale extract significantly increased TM3 cells proliferation activity compared to the untreated control. The relative mRNA expression levels of the cell proliferation-related genes Ki-67 and PCNA, anti-apoptotic gene Bcl-2, testosterone synthase genes StAR, P450scc, 3β-HSD, CYP17a1, and 17β-HSD, their relevant transcription factors genes Nur77 and SF-1, and upstream regulator gene CREB; as well as the relative protein expression levels of Bcl-2, P450scc, CYP17a1, and 17β-HSD were extremely significantly higher in Dendrobium officinale extract treated cells than those in the untreated control. The relative protein expression levels of Bax and Caspase-3, as well as the relative mRNA and protein expression levels of NF-κB were extremely significantly lower in Dendrobium officinale extract treated cells than those in the untreated control. The mass concentration of testosterone secreted by TM3 cells treated with Dendrobium officinale extract was (0.042±0.002) ng/mL, which was extremely significantly higher than that in the untreated control. In conclusion, 40 μg/mL Dendrobium officinale extract could enhance the synthesis of testosterone by regulating the expression of testosterone synthesis genes and their related transcription factors.