The edible mushroom Termitomyces is an agaric-type basidiomycete fungus that has a symbiotic relationship with fungus-growing termites. An understanding of the detailed development mechanisms underlying the adaptive responses of Termitomyces sp. to their growing environment is lacking. Here, we compared the transcriptome sequences of different Termitomyces sp. samples and link-stipe grown on fungus combs in situ and monocultured in vitro. The assembled reads generated 8052 unigenes. The expression profiles were highly different for 2556 differentially expressed genes (DEGs) of the treated samples, where the expression of 1312 and 1244 DEGs was upregulated in the Mycelium and link-stipe groups, respectively. Functional classification of the DEGs based on both Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed an expected shift in fungal gene expression, where stress response genes whose expression was upregulated in link-stipe may adaptively be involved in cell wall hydrolysis and fusion, pathogenesis, oxidation-reduction, transporter efflux, transposon efflux, and self/non–self-recognition. Urease has implications in the expression of genes involved in the nitrogen metabolism pathway, and its expression could be controlled by low-level nitrogen fixation of fungus combs. In addition, the expression patterns of eleven select genes on the basis of qRT-PCR were consistent with their changes in transcript abundance, as revealed by RNA sequencing. Taken together, these findings may be useful for enriching the knowledge concerning the Termitomyces adaptive response to in situ fungus combs compared with the response of monocultures in vitro.