Mitochondrial remodelling is a central feature of stem cell differentiation. However, little is known about the regulatory mechanisms during these processes. Previously, we found that a pharmacological inhibitor of glycogen synthase kinase-3α and -3β, CHIR-99021, initiates human adipose stem cell differentiation into human definitive endodermal progenitor cells (hEPCs), which were directed to differentiate synchronously into hepatocyte-like cells after further treatment with combinations of soluble factors. In this study, we show that CHIR-99021 promotes mitochondrial biogenesis, the expression of PGC-1α (also known as PPARGC1A), TFAM and NRF1 (also known as NFE2L1), oxidative phosphorylation capacities, and the production of reactive oxygen species in hEPCs. Blocking mitochondrial dynamics using siRNA targeting DRP1 (also known as DNM1L) impaired definitive endodermal differentiation. Downregulation of β-catenin (CTNNB1) expression weakened the effect of CHIR-99021 on the induction of mitochondrial remodelling and the expression of transcription factors for mitochondrial biogenesis. Moreover, CHIR-99021 decreased the expression of miR-19b-2-5p, miR-23a-3p, miR-23c, miR-130a-3p and miR-130a-5p in hEPCs, which target transcription factors for mitochondrial biogenesis. These data demonstrate that CHIR-99021 plays a role in mitochondrial structure and function remodelling via activation of the β-catenin signalling pathway and inhibits the expression of miRNAs during definitive endodermal differentiation.This article has an associated First Person interview with the first author of the paper.