Berberine, an important natural isoquinoline alkaloid from traditional Chinese medicine, is reported to exhibit multiple pharmacological properties, including anti-microbial, anti-diabetes, anti-obesity, anti-inflammatory and anti-carcinogenic activities. Although studies have shown that a wide range of carcinoma cells could be inhibited by berberine, few studies involved thyroid carcinoma. We therefore examined the effect of berberine on papillary thyroid carcinoma (PTC, the most common subtype) and anaplastic thyroid carcinoma (ATC, the most malignant and aggressive subtype). Three thyroid carcinoma cell lines with different aberrant genotypes and one normal thyroid cell line were selected, including C643 (ATC, with H-RAS mutation), OCUT1 (ATC, with BRAF and PIK3CA mutations), TPC1 (PTC, with RET/PTC1 rearrangement) and Htori3 (normal). We found that berberine inhibited the proliferation of C643, OCUT1 and TPC1 thyroid carcinoma cells in a dose- and time-dependent manner (p<0.01, compared with the control), while normal human thyroid cells showed less sensitivity to the cytotoxicity of berberine. Berberine-induced significant mitochondrial apoptosis, G0/G1 cell cycle arrest and inhibitive migration in thyroid carcinoma cells were also observed (p<0.05 or p<0.01, compared with the control). Increased Bax/Bcl-2, cleaved Caspase 3, P21 and decreased Cyclin E1, CDK2 and Vimentin were verified by western blot. Additionally, berberine caused markedly decreased p-AKT1 expression and disturbances in classic ERK MAPK as well as P38 and JNK MAPK pathways in diverse thyroid carcinoma cells. Therefore, berberine could modulate PI3K-AKT and MAPK signaling pathways in thyroid carcinoma cells, which leads to mitochondrial apoptosis, G0/G1 cell cycle arrest and suppressive migration. Berberine may represent a promising chemotherapy for the treatment of thyroid carcinoma.