<p>β-cell regeneration through the transdifferentiation of intra-islet cells presents a promising strategy for diabetes therapy. To identify the potential compound for induction of α-to-β cell transdifferentiation, we employed an in vivo screening using lineage trajectory analyses in zebrafish. We screened a library with 303 natural compounds from Chinese herbal medicine. We identified mosloflavone, a principal component of <i>Mosla soochouensis Matsuda</i>, which significantly induced the conversion of pancreatic α cells to β cells in zebrafish. Moreover, mosloflavone also converted α cells into β cells when β cells were extremely lost in zebrafish. Furthermore, mosloflavone administration increased the β-/α-cell ratio and β-cell mass, as well as improved glucose tolerance in HFD/STZ diabetic mice. Mechanistically, mosloflavone significantly increased the level of Pdx1 in α cells, both in zebrafish and mice. Hence, our study identified mosloflavone as an active compound derived from traditional Chinese medicine (TCM) that effectively induced the conversion of α cells to β cells, highlighting its potential to promote β cells regeneration as a therapeutic strategy for diabetes treatment.</p> Graphical abstract <p>Working model for Mosloflavone induced α-cell to β-cell conversion</p>