The widely used non-steroidal anti-inflammatory drugs (NSAIDs) are derivatives of the phytohormone salicylic acid (SA). SA is well known to regulate plant immunity and development, whereas there have been few reports focusing on the effects of NSAIDs in plants. Our studies here reveal that NSAIDs exhibit largely overlapping physiological activities to SA in the model plant Arabidopsis. NSAID treatments lead to shorter and agravitropic primary roots and inhibited lateral root organogenesis. Notably, in addition to the SA-like action, which in roots involves binding to the protein phosphatase 2A (PP2A), NSAIDs also exhibit PP2A-independent effects. Cell biological and biochemical analyses reveal that many NSAIDs bind directly to and inhibit the chaperone activity of TWISTED DWARF1, thereby regulating actin cytoskeleton dynamics and subsequent endosomal trafficking. Our findings uncover an unexpected bioactivity of human pharmaceuticals in plants and provide insights into the molecular mechanism underlying the cellular action of this class of anti-inflammatory compounds. Display omitted •NSAIDs exhibit striking activity in shaping Arabidopsis root development•NSAIDs interfere with auxin transport and endomembrane trafficking•NSAID treatments impair actin filament dynamics and endosomal mobility•NSAIDs directly target TWD1, suppressing its chaperone activity and actin dynamics Non-steroidal anti-inflammatory drugs (NSAIDs) are based on the phytohormone salicylic acid, but there are few studies investigating their bioactivities in plants. Tan et al. show that NSAIDs modulate Arabidopsis root development via directly binding to the immunophilin protein TWD1 and hence regulate its chaperone activity and endomembrane trafficking.