Dioscorea zingiberensis is the main plant source of diosgenin, a precursor for the production of steroid hormones used in the pharmaceutical industry. The extraction process of diosgenin from D. zingiberensis can generate high-acid and high-strength wastewater on a large scale and can threaten the environment. Bioengineering microorganisms to produce diosgenin is an effective way to avoid pollution. However, little is known about the genes that are involved in the biosynthesis of diosgenin. We obtained 85,010 unigenes (average length of 1142 bases) from the D. zingiberensis transcriptome through RNA-seq. A large number of unigenes (59,368; 69.83%) were annotated, and 2488 unigenes were assigned to 27 secondary-metabolite pathways. In our database, 66 unigenes encoding up to 40 key enzymes were found to be present in diosgenin biosynthesis pathways. In addition, we found 203 unigenes encoding CYP450 proteins and 47 unigenes encoding UGT proteins that may be involved in modifications of a downstream pathway. The expression patterns of key diosgenin biosynthesis genes were studied to identify the most important members of the enzyme family. These results add to the available genetic data of D. zingiberensis and lay the foundation for the further production of diosgenin using genetic engineering.