The production conditions of cordycepin, which is a kind of nucleoside analogue, were investigated by a surface culture using Cordyceps militaris NBRC 9787. And The effect of ferrous sulfate addition on production of cordycepin (3’ -deoxyadenosine) The optimal carbon/nitrogen ratio (w/w) was 2/1 when glucose was used as the carbon source. The lower medium depth resulted in a higher productivity over the present experimental range from 1.8 to 5.3 cm. Under the optimal conditions, the maximum cordycepin concentration in the culture medium reached 640 mg/l, and the maximum cordycepin productivity was 32 mg/(l•day). The results showed that the optimal addition condition was on day 0 with 1 g/L of ferrous sulfate, and the maximal amount of cordycepin reached 596.59±85.5 mg/L, about 70% higher than the control without ferrous sulfate addition. Meanwhile, the consumption of inosine 5 -monophosphate (IMP), a potential precursor of cordycepin, was decreased rapidly. Transcription levels of important genes encoding adenylosuccinate synthetase (purA), IMP cyclohydrolase (purH) and IMP dehydrogenase (guaB).