Adipogenesis relies on a tightly controlled cascade in which various factors respond in a sequential manner. It has been hypothesized that adipocyte differentiation is directed by a carefully timed activation of extracellular signal-regulated kinases (ERK1/2). Previous studies have demonstrated that ERK1/2 gene expression is controlled by the cytoplasmic ‘anchor’ called phosphoprotein enriched in astrocytes (PEA-15), regulating nuclear translocation of ERK1/21. Here, we reveal for the first time that PEA-15 regulates adipogenesis. Values are mean±S.E.M. and compared by ANOVA and unpaired t test. PEA-15 knockout (KO) mice and C57BL/6 (WT) mice were fed high fat diet (HFD, 42% kcal from fat) for 8 weeks (ethical approval by Ethics Review Board of University of Aberdeen). There was an increased body weight gain in PEA-15 KO compared to WT mice (PEA-15 KO-13.3±0.8g vs. WT-8.0±0.7g weight gain, p<0.001, n=8) and 50% greater mass of epididymal adipose tissue (eWAT) (PEA-15 KO-2.7±0.2g vs. WT-1.8±0.2g, p<0.05, n=4). Remarkably, serum triglyceride levels were reduced in PEA-15 KO mice (0.9±0.1 mg/mL vs. 1.6±0.3 mg/mL in WT, p<0.05, n=4). Despite the increased body weight, there was no additional hepatic lipid accumulation (TG assay) nor worsening of glucose intolerance (GTT assay). These changes in lipid handling could be explained by an increased efficiency in lipid storage in PEA-15-deficient adipose tissue, since adipocytes in eWAT sections were larger in PEA-15 KO mice (5.9±0.3μm2 vs. WT-4.4±0.5μm2, p<0.05, n=6). Interestingly, PEA-15 protein levels were increased in the adipose tissue of WT mice after HFD compared to WT mice on chow. Isolated pre-adipocytes from the eWAT of PEA-15 KO mice showed an increased initiation of adipogenesis followed by impaired maturation, as measured by protein levels of markers of adipocyte development. This suggests a previously uncharacterized and significant role for PEA-15 during adipocyte development. Confocal images of ERK1/2 immunostaining of adipocytes in PEA-15 KO eWAT showed that ERK1/2 is predominantly located in the nuclei compared to the cytoplasmic localization in WT eWAT. The concept of ERK1/2 translocation was confirmed in 3T3L1, in which differentiation led to ERK1/2 translocation. To unravel the regulatory mechanism of PEA-15, 3T3L1 adipocytes were transfected to knockdown (KD) PEA-15 gene expression. To determine whether PEA-15 KD led to increased downstream components of the ERK1/2 cascade, the activation of Elk1 was measured, being the transcription factor directly activated by ERK1/2. Indeed, Elk1 was constantly active in PEA-15 KD cells, as detected by EMSA (3.6±0.9 vs. control cells-0.4±0.2, p<0.05, n=3). This study demonstrates that PEA-15 protein deletion attenuates the HFD-associated lipid burden by increasing lipid storage capacity in adipocytes and preventing lipid overflow. Therefore, decreasing PEA-15 levels may initiate such favourable adiposity.