Plants' developmental plasticity plays a pivotal role in responding to environmental conditions. One of the most plastic plant organs is the root system. Different environmental stimuli such as nutrients and water deficiency may induce lateral root formation to compensate for a low level of water and/or nutrients. It has been shown that the hormone auxin tunes lateral root development and components for its signaling pathway have been identified. Using chemical biology, we discovered an Arabidopsis thaliana lateral root formation mechanism that is independent of the auxin receptor SCFTIR. The bioactive compound Sortin2 increased lateral root occurrence by acting upstream from the morphological marker of lateral root primordium formation, the mitotic activity. The compound did not display auxin activity. At the cellular level, Sortin2 accelerated endosomal trafficking, resulting in increased trafficking of plasma membrane recycling proteins to the vacuole. Sortin2 affected Late endosome/PVC/MVB trafficking and morphology. Combining Sortin2 with well-known drugs showed that endocytic trafficking of Late E/PVC/MVB towards the vacuole is pivotal for Sortin2-induced SCFTIR-independent lateral root initiation. Our results revealed a distinctive role for endosomal trafficking in the promotion of lateral root formation via a process that does not rely on the auxin receptor complex SCFTIR.