Isoleucine is one of the branched-chain amino acids (BCAAs) that are essential substrates for protein synthesis in all organisms. Although the metabolic pathway for isoleucine has been well characterized in higher plants, it is not known whether it plays a specific role in plant development. In this study, an Arabidopsis mutant, lib (low isoleucine biosynthesis), that has defects in both cell proliferation and cell expansion processes during root development, was characterized. The lib mutant carries a T-DNA insertion in the last exon of the OMR1 gene that encodes a threonine deaminase/dehydratase (TD). TD catalyses the deamination and dehydration of threonine, which is the first and also the committed step in the biosynthesis of isoleucine. This T-DNA insertion results in a partial deficiency of isoleucine in lib root tissues but it does not affect its total protein content. Application of exogenous isoleucine or introduction of a wild-type OMR1 gene into the lib mutant can completely rescue the mutant phenotypes. These results reveal an important role for isoleucine in plant development. In addition, microarray analysis indicated that the partial deficiency of isoleucine in the lib mutant triggers a decrease in transcript levels of the genes encoding the major enzymes involved in the BCAA degradation pathway; the analysis also indicated that many genes involved in the biosynthesis of methionine-derived glucosinolates are up-regulated.