Morchella is a genus of fungi with the ability to concentrate Cd both in the fruit-body and mycelium. However, the molecular mechanisms conferring resistance to Cd stress in Morchella are unknown. Here, RNA-based transcriptomic sequencing was used to identify the genes and pathways involved in Cd tolerance in Morchella spongiola. 7444 differentially expressed genes (DEGs) were identified by cultivating M. spongiola in media containing 0.15, 0.90, or 1.50 mg/L Cd 2þ . The DEGs were divided into six sub-clusters based on their global expression profiles. GO enrichment analysis indicated that numerous DEGs were associated with catalytic activity, cell cycle control, and the ribosome. KEGG enrichment analysis showed that the main pathways under Cd stress were MAPK signaling, oxidative phosphorylation, pyruvate metabolism, and propanoate metabolism. In addition, several DEGs encoding ion transporters, enzymatic/non-enzymatic antioxidants, and transcription factors were identified. Based on these results, a preliminary gene regulatory network was firstly proposed to illustrate the molecular mechanisms of Cd detoxification in M. spongiola. These results provide valuable insights into the Cd tolerance mechanism of M. spongiola and constitute a robust foundation for further studies on detoxification mechanisms in macrofungi that could potentially lead to the development of new and improved fungal bioremediation strategies.