Several studies have reported the effects of the consumption of various mushroom species on the testes in animal experimental models. Mushrooms, including enokitake mushrooms (Flammulina velutipes), and vegetables contain adenosine may affect testosterone production. Here, we aimed to elucidate the effects of enokitake and its active component, adenosine, on testosterone production in primary cultures of testicular cells in vivo using mice models and in vitro. The administration of enokitake ethanolic extract increased testosterone production in the cisplatin-impaired mouse model. The direct effect of mushroom extracts on testicular cells was examined and liquid chromatography–mass spectrometry analysis confirmed that the mushroom- and vegetable-induced increase in testosterone production mainly involved adenosine. Additionally, the administration of enokitake extract or adenosine to wet floor fatigue model mice promoted testicular testosterone production and enhanced Leydig cell function through insulin-like peptide three level upregulation. Structurally related compounds, including cordycepin, showed lower bioactivity than adenosine. This study showed that the ingestion of adenosine-containing mushrooms and vegetables may effectively increase testicular testosterone production. We conclude that mushrooms with a relatively high adenosine content, such as enokitake, may be useful against aging and fatigue.