Coumarin is a phytotoxic natural compound able to affect plant growth and development. Previous studies have demonstrated that this molecule at low concentrations (100 µM) can reduce primary root growth and stimulate lateral root formation, suggesting an auxin-like activity. In the present study, we evaluated coumarin’s effects (used at lateral root-stimulating concentrations) on the root apical meristem and polar auxin transport to identify its potential mode of action through a confocal microscopy approach. To achieve this goal, we used several Arabidopsis thaliana GFP transgenic lines (for polar auxin transport evaluation), immunolabeling techniques (for imaging cortical microtubules), and GC-MS analysis (for auxin quantification). The results highlighted that coumarin induced cyclin B accumulation, which altered the microtubule cortical array organization and, consequently, the root apical meristem architecture. Such alterations reduced the basipetal transport of auxin to the apical root apical meristem, inducing its accumulation in the maturation zone and stimulating lateral root formation.