Thrombocytopenia is closely linked with hemorrhagic diseases, for which induction of thrombopoiesis shows promise as an effective treatment. Polyphenols widely exist in plants and manifest antioxidation and antitumour activities. In this study, we investigated the thrombopoietic effect and mechanism of 3,3',4'-trimethylellagic acid (TMEA, a polyphenol in Sanguisorba officinalis L.) using in silico prediction and experimental validation. A KEGG analysis indicated that PI3K/Akt signalling functioned as a crucial pathway. Furthermore, the virtual molecular docking results showed high-affinity binding (a docking score of 6.65) between TMEA and mTOR, suggesting that TMEA might target the mTOR protein to modulate signalling activity. After isolation of TMEA, in vitro and in vivo validation revealed that this compound could promote megakaryocyte differentiation/maturation and platelet formation. In addition, it enhanced the phosphorylation of PI3K, Akt, mTOR, and P70S6K and increased the expression of GATA-1 and NF-E2, which confirmed the mechanism prediction. In conclusion, our findings are the first to demonstrate that TMEA may provide a novel therapeutic strategy that relies on the PI3K/Akt/mTOR pathway to facilitate megakaryocyte differentiation and platelet production.Copyright © 2021 Li, Jiang, Shen, Sun, Jiang, Zeng, Lin, Yue, Lai, Li, Wu, Wang, Qin, Huang, Mei, Yang and Wu.