Cordycepin, a natural adenosine derivative, exhibits multiple pharmacological effects on organisms. However, its distribution and metabolic characteristics have not been fully elucidated in vivo. In this study, ultra-high liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HRMS/MS) was used to investigate the pharmacokinetic characteristics and effects of cordycepin on endogenous adenosine and inosine. Microdialysis was used for real-time monitoring of unbound drug in brain and blood, whereas conventional tissue homogenate methods assessed distribution in various tissues. Results showed that the distribution pattern of cordycepin was as follows: kidney >
liver >
heart >
lung >
spleen >
brain. Cordycepin administration significantly altered the levels of adenosine and inosine in heart and liver. Synchronous microdialysis sampling for the pharmacokinetic profile indicated that cordycepin was rapidly consumed and 3'-deoxyinosine was generated as the main metabolite. The C