Background The principal manifestation of hyperuricemia is gout. Many drugs are in use nowadays to treat gout, but they are linked with multiple side effects. The present study observed berberine (from Chinese folk medicine) on serum and urinary uric acid levels in rats with potassium oxonate-induced hyperuricemia. Materials and methods Thirty-six adult healthy female Sprague Dawley rats were randomly divided into six groups of six rats each. To induce hyperuricemia, all the groups except Group A were given potassium oxonate (250 mg/kg) intraperitoneally on days 1, 3, and 7. Group A, the normal control group, was given normal saline for seven consecutive days intraperitoneally. Group C was administered allopurinol (5 mg/kg body weight) intraperitoneally, and Group D, E, and F were given berberine in doses of 0.75 mg/kg, 1.25 mg/kg, and 2.5 mg/kg body weight respectively intraperitoneally for seven consecutive days, one hour after the potassium oxonate injection. On zero, first, third, and seventh day of the experiment, blood and urine samples were taken to estimate the serum and urinary uric acid levels. On days zero and 7, serum uric acid was measured by cardiac puncture, while on days 1 and 3, it was measured by the tail prick method. The uric acid was measured by an enzymatic colorimetric method and creatinine by the Jaffe method. Fractional excretion of urate was also calculated. Results Berberine lowered serum uric acid levels in rats with potassium oxonate-induced hyperuricemia with highly significant results (p-value <0.001) in all three dosages. Berberine increased the urinary uric acid level and the fractional excretion of urate in a time-dependent manner in all three dosages. This effect was maximally shown by low dose berberine with a highly significant result (p-value <0.001). Conclusion Berberine successfully decreased the serum uric acid level of hyperuricemic rats by increasing the urinary uric acid level and fractional excretion of urate.