A RP-HPLC analytical method was developed for determining bergenin in rat urine, feces and tissues. The separation was performed on a C18 analytical column with methanol-water as the mobile phase at a flow rate of 0.8 mL/min. The column temperature was 40℃ and the eluate was monitored at 220 nm. The calibration curve for urine analysis showed a good linearity (r = 0.9989) over the range of 0.5-100 μg/mL, and the absolute recoveries were in the range of 90.0%-92.6%. A good linearity (r≥0.9996) for all tested tissues was obtained over the range of 0.3-150 μg/g except for lung, which had a linearity range of 0.5-150 μg/g. The absolute recoveries from tissues were higher than 63.9%. The method was successfully applied to determine the bergenin levels in several biological samples after rats were given bergenin formulation through tail vein injection. The results demonstrated that the amount of bergenin recovered from urine was about 35.36% of the total dose, and that from feces was less than 10%. Bergenin can be quickly and widely distributed in the rat, accumulating high levels in the kidney in comparison to all other tissues investigated. It was not distributed into the brain at any significant level owing to its hydrophilic property.