AbstractMetabolic programming is important for B cell fate, but the bioenergetic requirement for regulatory B (B_reg) cell differentiation and function is unknown. Here we show that B_reg cell differentiation, unlike non-B_reg cells, relies on mitochondrial electron transport and homeostatic levels of reactive oxygen species (ROS). Single-cell RNA sequencing analysis revealed that TXN, encoding the metabolic redox protein thioredoxin (Trx), is highly expressed by B_reg cells, unlike Trx inhibitor TXNIP which was downregulated. Pharmacological inhibition or gene silencing of TXN resulted in mitochondrial membrane depolarization and increased ROS levels, selectively suppressing B_reg cell differentiation and function while favoring pro-inflammatory B cell differentiation. Patients with systemic lupus erythematosus (SLE), characterized by B_reg cell deficiencies, present with B cell mitochondrial membrane depolarization, elevated ROS and fewer Trx⁺ B cells. Exogenous Trx stimulation restored B_reg cells and mitochondrial membrane polarization in SLE B cells to healthy B cell levels, indicating Trx insufficiency underlies B_reg cell impairment in patients with SLE.