Poria cocos (Schw.) Wolf, an important medicinal and food fungus, is well known in East Asia. Due to growing market demand, long cultivation period, and consumption of pine trunk during cultivation, developing alternative methods for producing P. cocos and/or its active components is of interest. In the present study, the effects of different culture methods on biomass and accumulation of four triterpenoids were investigated. The ethanol extract of fermented mycelium (EFM) was orally administered to rats. Urine output and concentrations of electrolytes (Na +, K +, and Cl –) were measured. Our results showed that mycelia grew better under continuous shaking culture condition (7.5 g DW·L –1), and higher triterpenoid levels were accumulated in two-stage culture (112 mg·L –1, 2.03%). The optimal starting time of static culture for triterpenoid yield was 4 th d after shaking culture. Single administration of middle and high dose of EFM significantly increased urine output, Na + and Cl – excretion, and Na +/K + ratio. These results suggested that ethanol extract of cultured mycelia showed significant diuretic activity in rats and two-stage culture of P. cocos could be an alternative way to produce mycelia and triterpenoids.