Griseofulvin was considered an effective agent for cancer therapy in past decades. Although the negative effects of griseofulvin on microtubule stability are known, the exact target and mechanism of action in plants remain unclear. Here, we used trifluralin, a well-known herbicide targeting microtubules, as a reference and revealed the differences in root tip morphology, reactive oxygen species production (ROS), microtubule dynamics, and transcriptome analysis betweenArabidopsistreated with griseofulvin and trifluralin to elucidate the mechanism of root growth inhibition by griseofulvin. Like trifluralin, griseofulvin inhibited root growth and caused significant swelling of the root tip due to cell death induced by ROS. However, the presence of griseofulvin and trifluralin caused cell swelling in the transition zone (TZ) and meristematic zone (MZ) of root tips, respectively. Further observations revealed that griseofulvin first destroyed cortical microtubules in the cells of the TZ and early elongation zone (EZ) and then gradually affected the cells of other zones. The first target of trifluralin is the microtubules in the root MZ cells. Transcriptome analysis showed that griseofulvin mainly affected the expression of microtubule-associated protein (MAP) genes rather than tubulin genes, whereas trifluralin significantly suppressed the expression of αβ-tubulin genes. Finally, it was proposed that griseofulvin could first reduce the expression of MAP genes, meanwhile increasing the expression of auxin and ethylene-related genes to disrupt microtubule alignment in root tip TZ and early EZ cells, induce dramatic ROS production, and cause severe cell death, eventually leading to cell swelling in the corresponding zones and inhibition of root growth.