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Baicalin is a major flavonoid compound purified from Scutellariae radix, which has been described as an herb in the Chinese Pharmacopoeia. Previous studies have suggested baicalin possessed extensive anti-inflammatory, anti-cancer, anti-viral properties. However, up to known, there have been no reports of safety and toxicity in the rats following oral administration of baicalin. In this present study, we showed the first evidence that treatment of baicalin (400, 800 and 1600 mg/kg/day) induced significantly kidney injury and fibrosis. The collagen synthesis and fibrosis-related protein expression were increased in the kidney of Sprague-Dawley (SD) rats after treatment with high doses of baicalin. We further investigated the potential molecular mechanism of baicalin-mediated renal fibrosis and revealed that baicalin activated the transforming growth factor-β (TGF-β)/Smad signaling pathway in a dose-dependent manner. Moreover, we also observed that baicalin induced Smad3 interaction with transcriptional coactivator p300 accompanying with increment of Smad3 acetylation. Our results may contribute to better understanding of the future pharmacological and toxicological studies of Scutellaria baicalensis Georgi and its active compounds on the human disease.High doses of baicalin induced kidney injury and fibrosis.High doses of baicalin activated TGF-β/Smad signaling pathway.High doses of baicalin increased the interaction of p300 and Smad3, and subsequent acetylation of Smad3.