ABSTRACTAims/IntroductionMetformin treatment for hyperglycemia in pregnancy (HIP) beneficially improves maternal glucose metabolism and reduces perinatal complications. However, metformin could impede pancreatic β cell development via impaired mitochondrial function. A new anti‐diabetes drug imeglimin, developed based on metformin, improves mitochondrial function. Here we examine the effect of imeglimin on β cell differentiation using human induced pluripotent stem cell (iPSC)‐derived pancreatic islet‐like spheroid (SC‐islet) models.Materials and MethodsHuman iPSCs are differentiated into SC‐islets by three‐dimensional culture with and without imeglimin or metformin. Differentiation efficiencies of SC‐islets were analyzed by flow cytometry, immunostaining, quantitative PCR, and insulin secretion assay. RNA sequencing and oxygen consumption rate were obtained for further characterization of SC‐islets. SC‐islets were cultured with proinflammatory cytokines, in part mimicking the uterus environment in HIP.ResultsMetformin perturbed SC‐islet differentiation while imeglimin did not alter it. Furthermore, imeglimin enhanced the gene expressions of β cell lineage markers. Maintenance of mitochondrial function and optimization of TGF‐β and Wnt signaling were considered potential mechanisms for augmented β cell maturation by imeglimin. In the presence of proinflammatory cytokines, imeglimin ameliorated β cell differentiation impaired by cytokines and metformin.ConclusionsImeglimin does not perturb differentiation of SC‐islet cells and rather enhances gain in β cell identity gene sets in contrast to metformin. This may lead to the improvement of in vitro β cell differentiation protocols.