Abscisic acid (ABA) is a sesquiterpenoid phytohormone involved in controlling plant root growth and development. Thymol, a monoterpene allelochemical, showed a potent phytotoxic effect in plants. It can rapidly inhibit seed germination and seedling growth. In this study, we employed a combination of transcriptome sequencing and validation methods from plant genetics and physiology to investigate the allelopathic inhibitory effects of thymol on the primary roots of Arabidopsis. We found that thymol affected the growth of Arabidopsis thaliana primary root in a dose-dependent manner, low concentration (10 mu M) generally enhances, and high concentration (150 mu M) inhibits. RNA sequencing analysis showed that a high concentration of thymol affected a series of biological processes and signaling transduction, including ABA biosynthesis, auxin polar transport, oxidative stress, root growth, and development. Exogenous ABA (10 mu M) enhanced the inhibitory effect of thymol on the primary root and the application of the ABA biosynthesis inhibitor Na2WO4 rescued this inhibitory effect. During this process, the content and distribution of auxin in the roots were significantly altered. The lengths of primary root and meristem of mutant abi1, abi2, and abi1 abi2, showed that ABI1 and ABI2 positively regulate the process of thymol inhibition of root growth. In summary, the allelopathic inhibitory of thymol on Arabidopsis thaliana primary root growth is mediated by ABA signaling pathway.