Pleurotus tuoliensis is a precious edible fungus with extremely high nutritive and medicinal value. The cultivation period of P. tuoliensis is longer than those of other Pleurotus species, which is mainly due to a longer mycelium physiological maturation period (30–60 days). Currently, the molecular processes underlying physiological maturation of the mycelium remain unclear. We performed a comparative transcriptomic analysis of immature and mature mycelia using RNA-seq. De novo transcriptome assembly resulted in identification of 17,030 unigenes. 451 differentially expressed genes—including those encoding nucleoside diphosphate kinase (NDPK), glycoside hydrolase family proteins, exopolygalacturonase, and versatile peroxidases—were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that nucleotide synthesis and energy metabolism are highly active during the physiological maturation of mycelia, and genes related to these pathways were significantly upregulated in mature mycelia. NDPK is predicted to be essential for mycelia maturation. Our findings contribute to a comprehensive understanding of mycelia maturation in a commercially important fungal species. Future efforts will focus on the function of NDPK and the mechanism by which it regulates mycelia maturation.