Caffeine contamination threatens ecosystems and human health, with conventional remediation methods facing limitations. This study identified Desarmillaria tabescens as a potent caffeine-degrading fungus, achieving efficient degradation under optimized conditions (malt extract medium, 900 mg/L caffeine, 28 °C, pH 8). HPLC analysis revealed key intermediates such as theobromine and 3-methylxanthine, confirming a branched catabolic pathway involving N-demethylation and C8 oxidation. Transcriptomic profiling identified nine consistently upregulated cytochrome P450 genes as core catalytic components, with three adjacent to a polyketide biosynthetic gene cluster potentially supporting oxidative reactions. A three-phase “Stress-Degradation-Homeostasis” regulatory model was proposed, coordinating detoxification, energy metabolism, and secondary metabolism. These findings advance understanding of fungal caffeine degradation mechanisms and provide valuable genetic resources for bioremediation and low-caffeine product development.

Caffeine contamination poses significant risks to both ecosystems and human health. Conventional remediation approaches, including adsorption and extraction, remain costly and lack substrate specificity, emphasizing the relevance of microbial degradation. The fungal species Desarmillaria tabescens demonstrated strong caffeine-degrading capacity under optimized conditions: malt extract medium supplemented with 900 mg/L caffeine at 28 °C and pH 8. HPLC and UPLC-MS/MS analyses revealed critical intermediates such as theobromine and 3-methylxanthine, indicating a branched catabolic route involving N-demethylation and C8 oxidation. Transcriptomic profiling identified stage-specific differential expression, with nine cytochrome P450 genes consistently upregulated. On this basis, a three-phase regulatory framework—“Stress-Degradation-Homeostasis”—was proposed, delineating the coordination of detoxification, energy metabolism, and secondary metabolic pathways. The elucidation of this mechanism not only advances understanding of D. tabescens caffeine metabolism but also provides genetic resources for bioremediation and the development of low-caffeine products.