Oct4 plays a crucial role in the regulation of self-renewal of embryonic stem cells (ESCs) and reprogramming of somatic cells to induced pluripotent stem cells. However, the molecular mechanisms underlying posttranslational regulation and protein stability of Oct4 remain unclear. Using affinity purification and mass spectrometry analysis, we identified Kap1 as an Oct4-binding protein. Silencing of Kap1 reduced the protein levels of Oct4 in ESCs, whereas the overexpression of Kap1 stimulated the levels of Oct4. In addition, Kap1 overexpression stimulated the self-renewal of ESCs and attenuated the spontaneous differentiation of ESCs in response to LIF withdrawal. Kap1 overexpression increased the stability of Oct4 by inhibiting the Itch-mediated ubiquitination of Oct4. Silencing of Kap1 augmented Itch-mediated ubiquitination and inhibited the stability of Oct4. We identified the lysine 133 (K133) residue in Oct4 as a ubiquitination site responsible for the Kap1-Itch-dependent regulation of Oct4 stability. Preventing ubiquitination at the lysine residue by mutation to arginine augmented the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. These results suggest that Kap1 plays a crucial role in the regulation of the pluripotency of ESCs and somatic cell reprogramming by preventing Itch-mediated ubiquitination and the subsequent degradation of Oct4.