Nitric oxide (NO) is a bioactive molecule that functions in numerous physiological and developmental processes in plants, including lateral root development. In this study, we used biochemical and genetic approaches to analyze the function of Arabidopsis thaliana mitogen-activated protein kinase 6 (MPK6) in the regulation of NO synthesis in response to hydrogen peroxide (H₂O₂) during lateral root development. In both mpk6 mutants studied, H₂O₂-induced NO synthesis and nitrate reductase (NR) activity were decreased dramatically. Furthermore, one NR isoform, NIA2, was required for the MPK6-mediated production of NO induced by H₂O₂. Notably, NIA2 interacted physically with MPK6 in vitro and in vivo and also served as a substrate of MPK6. Phosphorylation of NIA2 by MPK6 led to an increase in NR activity, and Ser-627 was identified as the putative phosphorylation site on NIA2. Phenotypical analysis revealed that mpk6-2 and mpk6-3 seedlings produce more and longer lateral roots than wild-type plants did after application of the NO donor sodium nitroprusside or H₂O₂. These data support strongly a function of MPK6 in modulating NO production and signal transduction in response to H₂O₂ during Arabidopsis root development.