Imazethapyr, a widely used herbicide, exhibits a long persistence in soils and can cause injury to rotational crops. Here, we discovered that imazethapyr inhibits primary root elongation in Arabidopsis by inhibiting cell division and expansion rather than damaging the organization of root meristem. Integration of transcriptomic and metabolomic analysis revealed that imazethapyr downregulated multiple genes related to cell wall loosening and modification, leading to increased cell wall thickness and inhibited cellular expansion in Arabidopsis roots. Furthermore, imazethapyr upregulated auxin biosynthesis and transport, resulting in enhanced auxin accumulation at root tips. Elevated auxin concentrations triggered apoplast alkalization and the inactivation of wall-loosening enzymes, further suppressing root growth. This research provides new insights into the molecular mechanism underlying imazethapyr phytotoxicity and offers potential strategies for developing crops that are better adapted to soils contaminated with imidazolinone herbicides.