Abstract: To determine the diuretic ingredients of Poria and reveal the underlying action mechanisms, multi-mode separation methods were employed to split the different fractions of Poria to yield Petroleum Ether Fraction (PEF), Ethyl Acetate Fraction (EAF), Water Eluated Fraction (WEF), Alcohol Eluated Fraction (AEF), Crude Polysaccharide Fraction (CPF) and Refined Polysaccharide Fraction (RPF). The rats were orally administered of the water decoction and different splitted fractions of Poria, purified water was used as control. Urine samples were collected to determine the concentration of Na+, K+ and Cl‾ and the blood samples were collected to determine the urine creatinine (Ucr), serum creatinine (Scr) and Na+-K+ ATPase. Furthermore, kidney tissues were also collected to test the Na+-K+ ATPase and Carbonic Anhydrase (CA). The results showed that the volume of urine and the concentration of Na+, K+ and Cl‾ in the 6 h urine samples were dramatically increased in the rats administered i.g., with water decoction and EAF as compared to the control group. Furthermore, the water decoction of Poria showed inhibitory effects on Na+-K+ ATPase in kidney while EAF showed inhibitory effects on the Na+-K+ ATPase action in both blood and kidney. Therefore, the EAF should be the diuretic fraction of Poria. The underlying diuretic action mechanisms of the EAF might be due to: (1) Increasing the release of Na+ and Cl‾ in urine and (2) Inhibiting the Na+-K+ ATPase activity in both blood and kidney.