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 Cmax values of cordycepin in the rat blood and brain after exposure (10 mg/kg, i.p.) were 7.8 and 5.4 ng/mL, respectively. Mean residence time in blood and brain was 102.2 and 137.0 min, respectively. Inhibition of adenosine deaminase by racemic 9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA) enhanced cordycepin levels in the blood. This work provides a solid basis for understanding the metabolism of cordycepin and its pharmacological effects.