Drought is an important abiotic factor constricting crop production globally. Although the role of JAZ proteins in regulating jasmonic acid signaling and plant responses to environmental stress is well documented, their specific functions and underlying mechanisms in plants remains little known. In this study, JAZ proteins in barley were thoroughly analyzed, revealing a total of 11 members classified into three phylogenetic subgroups. HvJAZ2, based on its distinct expression patterns, is considered as a key candidate gene for regulating drought tolerance in barley. Using the HvJAZ2 knockout mutants, we revealed that the gene negatively regulates drought tolerance by inhibiting barley root growth. Notably, the jaz2 mutants up-regulated expression of root development genes, including SHR1, PLT1, PLT2, and PLT6. plt2 and plt1/plt2 mutants exhibited suppressed root development and reduced drought tolerance. Analysis of interactions between HvJAZ2 and other proteins showed that HvJAZ2 is not directly interacted with HvPLT1/2/6, but interacts with some other proteins. BIFC and LCA assays further confirmed the nuclear interaction between HvJAZ2 and HvMYC2. Y1H and Dual-Luciferase experiments demonstrated that HvMYC2 can bind to and activates the HvPLT2 promoter. In summary, HvJAZ2 negatively regulates root development and drought tolerance in barley by suppressing HvPLT2 expression through interacting with HvMYC2.