The 14-3-3 protein is a kind of evolutionary ubiquitous protein family highly conserved in eukaryotes. Initially, 14-3-3 proteins were reported in mammalian nervous tissues, but in the last decade, their role in various metabolic pathways in plants established the importance of 14-3-3 proteins. In the present study, a total of 2214-3-3genes, also called general regulatory factors (GRF), were identified in the peanut (Arachis hypogaea) genome, out of which 12 belonged to the ε group, whereas 10 of them belonged to the non- ε-group. Tissue-specific expression of identified14-3-3genes were studied using transcriptome analysis. The peanutAhGRFigene was cloned and transformed intoArabidopsis thaliana. The investigation of subcellular localization indicated thatAhGRFiis localized in the cytoplasm. Overexpression of theAhGRFigene in transgenicArabidopsisshowed that under exogenous 1-naphthaleneacetic acid (NAA) treatment, root growth inhibition in transgenic plants was enhanced. Further analysis indicated that the expression of auxin-responsive genesIAA3,IAA7,IAA17, andSAUR-AC1was upregulated andGH3.2andGH3.3were downregulated in transgenic plants, but the expression ofGH3.2,GH3.3, andSAUR-AC1showed opposite trends of change under NAA treatment. These results suggest thatAhGRFimay be involved in auxin signaling during seedling root development. An in-depth study of the molecular mechanism of this process remains to be further explored.