The aim of this work was to investigate the effect of the whole-body deletion of p27 on the activity of brown adipose tissue and the susceptibility to develop obesity and glucose homeostasis disturbances in mice, especially when subjected to a high fat diet. p27 knockout (p27(-/-)) and wild type (WT) mice were fed a normal chow diet or a high fat diet (HFD) for 10-weeks. Body weight and composition were assessed. Insulin and glucose tolerance tests and indirect calorimetry assays were performed. Histological analysis of interscapular BAT (iBAT) was carried out, and expression of key genes/proteins involved in BAT function were characterized by qPCR and Western blot. iBAT activity was estimated by F-18-fluorodeoxyglucose ((18)FDG) uptake with microPET. p27(-/-) mice were more prone to develop obesity and insulin resistance, exhibiting increased size of all fat depots. p27(-/-) mice displayed a higher respiratory exchange ratio. iBAT presented larger adipocytes in p27(-/-) HFD mice, accompanied by downregulation of both Glut1 and uncoupling protein 1 (UCP1) in parallel with defective insulin signalling. Moreover, p27(-/-) HFD mice exhibited impaired response to cold exposure, characterized by a reduced iBAT (18)FDG uptake and difficulty to maintain body temperature when exposed to cold compared to WT HFD mice, suggesting reduced thermogenic capacity. These data suggest that p27 could play a role in BAT activation and in the susceptibility to develop obesity and insulin resistance.