Abstract The presence of adult pluripotent stem cells and the amazing regenerative capabilities make planarian flatworms an extraordinary experimental model to assess in vivo the effects of substances of both natural and synthetic origin on stem cell dynamics. This study focuses on the effects of chelidonine, an alkaloid obtained from Chelidonium majus. The expression levels of molecular markers specific for stem or differentiated cells were compared in chelidonine-treated and control planarians. The use of these markers demonstrates that chelidonine produces in vivo a significant anti-proliferative effect on planarian stem cells in a dose-dependent fashion. In response to chelidonine treatment mitotic abnormalities were also observed and the number of cells able to proceed to anaphase/telophase appeared significantly reduced with respect to the controls. Our results support the possibility that chelidonine acts on cell cycle progression by inhibition of tubulin polymerization. These studies provide a basis for preclinical evaluation in vivo of the effects of chelidonine on physiologically proliferating stem cells.