Colchicine (CAS 64-86-8) is considered to have a hepatoprotective effect and play a role in biliary excretion. 17alpha-Ethynylestradiol (EE) (5 mg/kg, subcutaneously, daily, for 5 days) causes intrahepatic cholestasis by reducing both the influx and efflux of bile acid in hepatocytes, resulting in a decrease in bile flow. The objective of this study was to evaluate whether colchicine has any effect on EE-induced cholestasis. The effects of colchicine treatment on EE-induced cholestasis in rats for 5 consecutive days were evaluated. The serum components and enzymatic activity were assayed. In addition, the bile flow and biliary excretion were determined. Furthermore, western blot analysis was used to measure the expression of farnesoid X receptor (FXR), bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), and cholesterol 7alpha-hydroxylase (CYP7A1). Colchicine not only significantly inhibited the elevation of cholestasis-related serum components and enzyme activity but also significantly attenuated the decrease of the bile flow and biliary excretion. Colchicine also remarkably increased the hepatic expression of FXR, BSEP and MRP2, but decreased that of CYP7A1. Our data indicates that colchicine treatment attenuated EE-induced cholestasis in rats, most likely by promoting bile flow and biliary excretion, and reduced the synthesis of bile acids.