Auricularia cornea is a widely prized basidiomycetous mushroom with culinary and medicine value, which is cultivated artificially on a large scale in China. However, little attention has been paid to the differences in metabolic profiles under different pH growth conditions. In the present study, weakly acidic and weakly alkaline artificial substrates were developed and used for the cultivation of A. cornea, and the metabolic profiles of its fruiting bodies were determined using ultra performance liquid chromatography-tandem mass spectrometry. The results show that the weakly alkaline substrate environment promoted mycelial growth, increased body surface area, and improved yield and transformation efficiency, but attenuated metabolite accumulation by A. cornea. A total of 412 different metabolites were identified in negative and positive ion mode, of which 99 had significantly different amounts and covered 51 metabolic pathways. Principal component analysis and orthogonal patrial least squares discriminant analysis showed clear separation between two treatments, indicating different metabolic profiles. The different metabolites mainly included seven chemical categories, including amino acids and derivatives, nucleotides and derivatives, phenolic acids, organic acids, lipids, flavonoids and alkaloids. This study revealed the biological significance of these metabolites, which could be useful for further unexplored compounds and possible biological functions of A. cornea. © 2023, Canadian Science Publishing. All rights reserved.