Weight loss and regain are associated with profound cytoskeleton changes in collapsing and re-expanding adipocytes. These drastic changes in the adipose tissue may drive proinflammatory signalling and fibrotic processes during weight regain. It remains unclear how adipose tissue re-expansion affects adipose tissue function and thereby the comorbidities related to obesity. The aim of this study was to characterize structural and metabolic changes in subcutaneous adipose tissue after weight loss and regain in diet-induced obesity (DIO) mice. DIO mice underwent calorie restriction to achieve a 25% weight loss and then returned to ad libitum high fat diet feeding to regain weight. Inguinal adipose tissue was collected and analysed from five groups of mice during the experiment: before high fat diet feeding (Lean) , before weight loss (DIO 1) , after weight loss (WL) , after 3 weeks of weight regain (WR) and age-matched ad libitum DIO mice (DIO 2) . We found that WR group had lower weight and fat mass compared to DIO2 group (31.9±1.6 vs. 23.9±1.9 %) and lower basal lipolysis measured ex-vivo. We detected a larger population of small adipocytes in WR mice compared to DIO2 group, resulting in smaller average adipocyte size (81.6±1.4 vs. 52.5±0.9 µm) and increased expression of cell renewal markers Cidea, Antrx1 and Prkaa in adipose tissue, indicating activation of adipogenesis. Expression of cytokines Il2, Ccl19, Cdc3 in adipose tissue in WR compared to DIO was reduced as well as plasma levels of cytokines Il2, KC/GRO and Inf-γ. In conclusion, our study does not support that increased adipogenesis in weight regain results in accelerated fat accumulation in mice. Weight regain is accompanied by reduced systemic and adipose inflammation. This suggests in general a beneficial effect of regain-induced adipogenesis resulting in potentially healthier adipose tissue in mice. Weather these findings represent the human situation with weight loss and regain is unknown and is currently under investigation. Disclosure M. K. Gerstenberg: Employee; Novo Nordisk. R. E. Kuhre: Employee; Novo Nordisk. N. Petersen: Employee; Novo Nordisk A/S. A. Bookout: Employee; Novo Nordisk. C. M. Castorena: Employee; Novo Nordisk. L. Torz: Other Relationship; Novo Nordisk A/S. T. H. Dovmark: None. S. Lundh: Employee; Novo Nordisk A/S. S. H. Madsen: None. B. S. Andersen: Employee; Novo Nordisk A/S. T. A. Pedersen: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S.