Objectives: Human population studies link sequence variants near the allograft inflammatory factor (Aif)-1 locus with obesity, adipose inflammation, and diabetes, conditions strongly associated with cardiovascular disease. We assessed possible causal roles of Aif-1 in these conditions using mouse models. Hypothesis: Aif-1 promotes obesity, adipose inflammation, and glucose intolerance. Methods: We generated Aif-1-deficient ( aif-1 -/- ) mice and fed them a normal or high fat diet (ND or HFD, respectively) for 16 weeks. Body composition was analyzed by microCT scan, tissues by macroscopic and microscopic methods to assess morphology, and gene expression by qRT-PCR. Metabolic activity was measured in metabolic chambers. Wt littermates served as controls. Results: Aif-1 was expressed in visceral, subcutaneous, and brown adipose tissues (VAT, SAT, and BAT, respectively). Aif-1 -/- mice showed less HFD-induced gain in body weight ( wt vs. aif-1 -/- ; 20 vs. 7.5g, p≤0.01) and fat mass ( wt vs. aif-1 -/- ; 21 vs.10g, p≤0.001), while lean mass did not change. We also found reduced tissue mass in VAT ( wt vs. aif-1 -/- ; 1.7 vs. 0.7g, p≤0.01), SAT ( wt vs. aif-1 -/- ; 2.5 vs. 0.4, p ≤0.0001) and BAT ( wt vs. aif-1 -/- ; 0.19 vs. 0.07, p≤0.01), with smaller adipocytes in both VAT and SAT. In addition, reduced lipid accumulation in BAT and liver was seen in aif-1 -/- mice on HFD compared to wt controls. Interestingly, aif-1 -/- mice showed increased metabolic activity (energy expenditure, wt vs. aif-1 -/- ; 14 vs.16 Kcal/kg/h, p≤0.05), while physical activity was unchanged. At the molecular level, aif-1 -/- mice showed increased expression of uncoupling protein-1 in BAT, SAT, and VAT, as well as its co-activators PGC1α and PRDM16 in BAT and SAT. These findings suggest increased brown and beige adipogenesis in BAT and SAT, respectively. Compared to controls, aif-1 -/- mice were also more glucose tolerant and insulin-sensitive on either ND or HFD, and this correlated with macrophage polarization toward an M2 vs. M1 phenotype in VAT. Conclusion: Our data suggest that Aif-1 deficiency prevents HFD-induced obesity by enhancing metabolic activity via increased brown or beige adipocyte programs, which reduces inflammation and promotes glucose tolerance and insulin sensitivity.