Objective: We investigated the in vitro and in vivo effects of IM156 on overactive B cells stimulated by the TLR9 agonist CpG oligodeoxynucleotide (ODN). Using RNA sequencing (RNA-seq), we analyzed the B-cell transcriptome expression, identifying the major molecular pathways affected by IM156 in vivo. We also evaluated the anti-inflammatory effects of IM156 in NZB/W F1 mice. Methods: We demonstrated that CD19+ B cells exhibited higher mitochondrial mass and mitochondrial membrane potential than T cells and were more susceptible to IM156-mediated oxidative phosphorylation (OXPHOS) inhibition (P < 0.01 or P < 0.001; N = 3–6). In vivo, IM156 inhibited OXPHOS, cell cycle progression, plasmablast differentiation, and activation marker levels in ODN-stimulated mouse spleen B cells. Interestingly, IM156 treatment significantly increased overall survival (P < 0.05; N = 3–9) and reduced proteinuria (P < 0.05 or P < 0.01; N = 3–9) in lupus-prone NZB/W F1 mice. Our data indicate that IM156 suppressed the mitochondrial membrane potentials of activated B cells in mice, which contributed to mitigation of lupus activity. Results: IM156 may represent a therapeutic alternative for autoimmune diseases mediated by B cell hyperactivity. Conclusions: Objective: Current treatment strategies for autoimmune diseases, including systemic lupus erythematosus, may not sufficiently control the aberrant metabolism in B cells. To address this concern, we investigated a biguanide derivative, IM156, as a potential regulator for B cell metabolism. Methods: We investigated the in vitro and in vivo effects of IM156 on overactive B cells stimulated by the TLR9 agonist CpG oligodeoxynucleotide (ODN). Using RNA sequencing (RNA-seq), we analyzed the B-cell transcriptome expression, identifying the major molecular pathways affected by IM156 in vivo. We also evaluated the anti-inflammatory effects of IM156 in NZB/W F1 mice. Results: We demonstrated that CD19+ B cells exhibited higher mitochondrial mass and mitochondrial membrane potential than T cells and were more susceptible to IM156-mediated oxidative phosphorylation (OXPHOS) inhibition (P < 0.01 or P < 0.001; N = 3–6). In vivo, IM156 inhibited OXPHOS, cell cycle progression, plasmablast differentiation, and activation marker levels in ODN-stimulated mouse spleen B cells. Interestingly, IM156 treatment significantly increased overall survival (P < 0.05; N = 3–9) and reduced proteinuria (P < 0.05 or P < 0.01; N = 3–9) in lupus-prone NZB/W F1 mice. Our data indicate that IM156 suppressed the mitochondrial membrane potentials of activated B cells in mice, which contributed to mitigation of lupus activity. Conclusions: IM156 may represent a therapeutic alternative for autoimmune diseases mediated by B cell hyperactivity.