MADS-box transcription factors play crucial roles in regulating development processes and biosynthesis of secondary metabolites in eukaryotes. However, the role of MADS-box transcription factors vary among fungal species, and their function remains unclear in the medicinally and economically important fungus Ganoderma lucidum. In this study, we characterized a MADS-box gene, GlMADS1, in G. lucidum. Analyses using quantitative real-time polymerase chain reaction (qRT-PCR) showed that GlMADS1 expression levels were up-regulated from the mycelia to the primordia stage. In order to further evaluate the effect of MADS-box transcription factors on secondary metabolism, we utilized RNA interference (RNAi) to silence GlMADS1 in G. lucidum. Ganoderic acid (GA) and flavonoid contents were enhanced in GlMADS1-silenced strains, suggesting that GlMADS1 negatively regulates GA and flavonoid accumulation.