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Cancer stem cells (CSCs) play a key role in the development and progression of colorectal cancer (CRC), but the influence of triiodothyronine (T3) on the biological regulation of CSCs remains unclear. In the present study, it was reported that T3 exerts significant impact on CSCs of two CRC cell lines cultured in the form of colonospheres. It was observed that the incubation of colonospheres with T3 decreased the viability, proliferative and spherogenic potential of cancer cells (P0.05). In addition, increased apoptotic rate of CRC cells treated with T3 was revealed. Furthermore, T3‑treated colonospheres were more likely to move into silenced pool in G0/G1 phase of the cell cycle. The smaller sizes of colonospheres observed after the treatment with T3 confirmed this conclusion. T3 could lower the proportion of primitive cells which supply the pool of proliferating cells within spheres. Thyroid receptors THRα1 and THRβ1 and two deiodinases (DIO2 and DIO3) were affected by T3 in manner depended on clinical stage of cancer and CRC cell line used for analysis. In summary, the present study uncovered a novel function of thyroid hormones signaling in the regulation of the CSCs of CRC, and these findings may be useful for developing novel therapies by targeting thyroid hormone functions in CRC cells.