Tremella fuciformis is an edible and medicinal white jelly mushroom. It has a life cycle that interacts with its companion fungus Annulohypoxylon stygium, both in natural conditions and artificial cultivation. RNA sequencing (RNA-Seq) was used to study the interaction between T. fuciformis and A. stygium by constructing 5 libraries, including the individual T. fuciformis mycelium (1), the T. fuciformis mycelium after interaction with A. stygium (2), the dual mycelia after interaction (3), the A. stygium mycelium after interaction with T. fuciformis (4), and the individual A. stygium mycelium (5). 33.4 G data and 46,871 Unigenes were generated from de novo splicing. For identification of differentially expressed genes (DEGs) related to interaction, we analyzed the expression data of DEGs1-vs-2 ∩ DEGs1-vs-3, and DEGs5-vs-4 ∩ DEGs5-vs-3. DEGs1-vs-2 ∩ DEGs1-vs-3, and DEGs5-vs-4 ∩ DEGs5-vs-3 data showed 614 DEGs and 1537 DEGs, respectively. The 614 DEGs for T. fuciformis and 1537 DEGs for A. stygium were analyzed by GO annotation and were assigned to biology process, cell composition, and molecular functions. The DEGs were used to match the KEGG database. In T. fuciformis, the pathways are primarily enriched various amino acids metabolism, pentose and glucuronate interconversions. In A. stygium, the pathways are primarily enriched in the biosynthesis of secondary metabolites, biosynthesis of antibiotics, starch and sucrose metabolism. The expression patterns of DEGs determined by qRT-PCR were consistent with those obtained by RNA-Seq, thus validating the reliability of our RNA-Seq data. Future studies of the functions of these interesting genes will be helpful to understand the mechanisms by which T. fuciformis interacts with A. stygium. This will also provide a reference for other research on interacting microorganisms.