Background: Ophiocordyceps sinensis (O. sinensis), is an entomopathogenic fungus renowned for its medicinal properties, thriving in the frigid and high-altitude regions of the Qinghai-Tibet plateau. Given the limited availability of wild resources and the increasing recognition of their medicinal value, the cultivation of O. sinensis was initiated. However, there is a paucity of research investigating the disparities in their respective qualities. In this study, untargeted metabolomics and proteomes were used to reveal the quality and mechanism differences between wild and cultivated O. sinensis. Results: The key representative components such as polysaccharide, crude protein, adenosine, and mannitol contents of wild O. sinensis were higher than those of cultivated O. sinensis. Due to the diversity in ecological environments, wild O. sinensis exhibits higher levels of LysoPA (0:0/16:0) and LysoPC (18:1(11Z)/0:0), whereas cultivated O. sinensis shows significantly elevated levels of 5-aminopentanal and succinic acid. Additionally, starch and sucrose metabolism, sphingolipid metabolism, and nucleotide metabolism pathways are significantly enriched with differentially expressed proteins (DEPs) and metabolites (DEMs). Correlation analysis revealed that different habitats altered 47 pathways shared between the proteome and metabolome, including carbohydrate metabolism, lipid metabolism, amino acid metabolism, metabolism of other amino acids, and energy metabolism. Especially, beta-glucosidase and alpha-galactosidase play crucial roles in the synthesis and metabolism of carbohydrates and amino acids. The different expression of polyamine oxidase in arginine and proline metabolism, suggests significant differences between wild and cultivated O. sinensis in ammonification and nitrogen balance maintenance, which may lead to quality disparities. Conclusions: In conclusion, these findings offer a novel perspective on the research advancements and applications of O. sinensis and serve as a reference for the targeted development of cultivated O. sinensis.