AIMS Argonaute2 (Ago2) plays a fundamental role in microRNA-mediated gene regulation through its intrinsic endonuclease activity. In this study we demonstrate the novel functions and molecular mechanisms by which nuclear Ago2 directly regulates HSP (heat shock protein) 60 expression and stem cell self-renewal. HSP60 is a crucial regulator of ROS (reactive oxygen species), senescence, and apoptotic cell death in several tissues and cell types. RESULTS HSP60 is regulated via inactivation of p38/JNK and p53 and binds directly to the regulatory regions of the TERT, c-myc, GPx3, p53, and STAT3 genes. Using HSP60 CHIP-PCR experiments, we show that HSP60 binds directly to the Oct4 and Nanog genes and directly regulates Oct4 and other stemness genes involved in human adipose tissue-derived stem cell (hATSC) differentiation. HSP60 also positively regulates ROS-scavenging factors, including GPx3 and TXNL1, which directly modulate cytosolic ROS in hATSCs. Moreover, our study shows that Oct4 regulates HSP60 expression and controls hATSC survival and self-renewal after binding to the HSP60 gene. Furthermore, HSP60-mediated regulation of Oct4 contributes to neuronal and endodermal β-cell differentiation of hATSCs in vitro and in vivo and downregulates mesoderm-specific gene expression. INNOVATION AND CONCLUSION We show that increased levels of Ago2 or HSP60 effectively induce nuclear localization of HSP60, which directly controls Oct4, c-Myc, p53, TERT, and STAT3 for transdifferentiation programs. Collectively, we suggest a novel model in which nuclear Ago2 controls HSP60 in hATSCs.