Murine protein serine-threonine kinase 38 (MPK38)/maternal embryonic leucine zipper kinase (MELK), an AMP-activated protein kinase (AMPK)-related kinase, has previously been shown to interact with p53 and to stimulate downstream signaling. p21, a downstream target of p53, is also known to be involved in adipocyte and obesity metabolism. However, little is known about the mechanism by which p21 mediates obesity-associated metabolic adaptation. Here, we identify MPK38 as an interacting partner of p21. p21 and MPK38 interacted through the cyclin-dependent kinase (CDK) binding region of p21 and the C-terminal domain of MPK38. MPK38 potentiated p21-mediated apoptosis and cell cycle arrest in a kinase-dependent manner by inhibiting assembly of CDK2-cyclin E and CDK4-cyclin D complexes via induction of CDK2-p21 and CDK4-p21 complex formation and reductions in complex formation between p21 and its negative regulator mouse double minute 2 (MDM2), leading to p21 stabilization. MPK38 phosphorylated p21 at Thr55, stimulating its nuclear translocation, which resulted in greater association of p21 with peroxisome proliferator-activated receptor γ (PPARγ), preventing the PPARγ transactivation required for adipogenesis. Furthermore, restoration of p21 expression by adenoviral delivery in diet-induced obese mice ameliorated obesity-induced metabolic abnormalities in a MPK38 phosphorylation-dependent manner. These results suggest that MPK38 functions as a positive regulator of p21, regulating apoptosis, cell cycle arrest, and metabolism during obesity.