Oral immunotherapy (OIT) can successfully desensitize many peanut-allergic subjects, but clinical tolerance diminishes over time on discontinuation, or low-dose maintenance, of peanut. Therefore, to improve the efficacy and sustainability of such therapy, we sought to identify biomarkers and clinical tools that can predict therapeutic outcomes and monitor treatment responses. We evaluated whether basophil activation in whole blood, and plasma levels of peanut-specific immunoglobulins, are useful biomarkers for peanut OIT. We longitudinally measured, before, during, and after OIT, basophil activation in whole blood ex vivo in response to peanut stimulation, and peanut-specific IgE (sIgE) and peanut-specific IgG4 (sIgG4), in a large, single-site, double-blind, randomized, placebo-controlled, phase 2 peanut OIT study. We compared basophil responsiveness and peanut-specific immunoglobulins between those who were clinically reactive and those who were tolerant to peanut oral challenges. Peanut OIT significantly decreased basophil activation, peanut sIgE, Ara h 1, Ara h 2, and Ara h 3 IgE levels, and sIgE/total IgE, but increased sIgG4/sIgE. Participants who became reactive to 4 g of peanut 13 weeks off active OIT exhibited higher peanut-induced basophil activation ex vivo and higher peanut sIgE levels and sIgE/total IgE, but lower sIgG4/sIgE. Notably, participants entering the study with low basophil responsiveness were more likely to achieve treatment success. Substantial suppression of basophil activation was required to maintain long-term clinical tolerance after peanut OIT. Assessments of peanut-induced basophil activation and peanut-specific immunoglobulins can help to predict treatment outcomes, and to differentiate transient desensitization versus sustained unresponsiveness after OIT. Copyright © 2019 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.