Leukemia inhibitory factor (LIF), an indispensable bioactive protein that sustains self-renewal and pluripotency in stem cells, is vital for mouse embryonic stem cell (mESC) culture. Extensive research is conducted on reliable alternatives for LIF as its clinical application in stable culture and large-scale expansion of ESCs is limited by its instability and high cost. However, few studies have sought to replace LIF with nanoparticles to provide a xeno-free culture condition. MgAl-LDH (layered double hydroxide) nanoparticles can partially replace LIF in maintaining pluripotency of mESCs; however, the requirement and tolerance for aluminum ions in mice are far lesser than those of iron ions. Hence, MgFe-LDH nanoparticles are selected for this study. MgFe-LDH is superior to MgAl-LDH in maintaining self-renewal and pluripotency of mESCs, in the absence of LIF and mouse embryonic fibroblast. Furthermore, combined transcriptomic and proteomic analysis confirms that MgFe-LDH can activate the LIF receptor (LIFR)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B(AKT), LIFR/JAK/janus kinase (JAK)/signal transducer and activator of transcription 3(STAT3), and phospho-signal transducer and activator of transcription 3(p-STAT3)/ten-eleven translocation (TET) signaling pathways, while the extra Fe provided by MgFe-LDH would also enhance TET1/2 abundance thus affecting the TET1/2 regulated pluripotency related marker expression and TET1/2 meditated DNA demethylation. These results suggest that MgFe-LDH nanoparticles can thus be used as an affordable and efficient replacement for LIF in mESC cultivation.