<p>Mesenchymal stem cells (MSCs) have many uses in tissue engineering and clinical applications. However, maintaining their stemness during in vitro expansion is challenging. We previously found that Krüppel-like factor 2 (KLF2) plays a crucial role in maintaining the stemness of MSCs. In this study, KLF2 was revealed to be closely linked to mitochondrial oxidative phosphorylation (OXPHOS), and impaired KLF2 expression in MSCs led to mitochondrial dysfunction that ultimately resulted in the loss of stemness. Moreover, decreased KLF2 expression was associated with reduced expression of the mitochondrial electron transport chain components, particularly the accessory subunit of complex I, NDUFC1. Further study demonstrated that KLF2 transcriptionally regulates NDUFC1 expression by binding to its promoter region. In addition, NDUFC1 knockdown largely phenocopied KLF2 knockdown in mitochondrial dysfunction and loss of stemness, and these phenotypes were partially rescued by NDUFC1 overexpression. Taken together, we reveal that KLF2 critically maintains MSCs stemness by transcriptionally promoting the expression of mitochondrial electron transport chain components such as NDUFC1, and KLF2/NDUFC1 axis-regulated mitochondrial oxidative phosphorylation may serve as a novel therapeutic target for improving MSCs stemness.</p>