Land plants co-speciate with a diversity of continually expanding plant specialized metabolites (PSMs) and root microbial communities (microbiota). Homeostatic interactions between plants and root microbiota are essential for plant survival in natural environments. A growing appreciation of microbiota for plant health is fuelling rapid advances in genetic mechanisms of controlling microbiota by host plants. PSMs have long been proposed to mediate plant and single microbe interactions. However, the effects of PSMs, especially those evolutionarily new PSMs, on root microbiota at community level remain to be elucidated. Here, we discovered sesterterpenes in Arabidopsis thaliana, produced by recently duplicated prenyltransferase-terpene synthase (PT-TPS) gene clusters, with neo-functionalization. A single-residue substitution played a critical role in the acquisition of sesterterpene synthase (sesterTPS) activity in Brassicaceae plants. Moreover, we found that the absence of two root-specific sesterterpenoids, with similar chemical structure, significantly affected root microbiota assembly in similar patterns. Our results not only demonstrate the sensitivity of plant microbiota to PSMs but also establish a complete framework of host plants to control root microbiota composition through evolutionarily dynamic PSMs.