Adipocyte browning is a potent therapeutic strategy for obesity and metabolic disorders. We previously reported that miR-494 served as a negative regulator of mitochondrial biogenesis during browning in cultured adipocytes. However, the in vivo evidence has not been provided. In this study, we examined the role of miR-494 in adipocyte browning and aging using miR-494 knockout (KO) mice. KO mice were generated using CRISPR/Cas9 system. KO mice had equivalent energy expenditure, physical activity, food intake and body weight compared to wild-type (WT) mice; had lower resting respiratory quotient (0.95±0.006 vs 0.91±0.004), less visceral fat (16.3±1.08 vs 13.4±0.96 g/kg body weight) and 3.7-fold increased expression of Ucp1 in subcutaneous fat. In addition, KO mice showed cold tolerance during 3 hours exposure to 4ºC and increased oxygen consumption than WT mice after administration of a β3 agonist in thermoneutral condition, indicating an increase in non-shivering thermogenesis. In adipose tissues of KO mice, increased expression of Pgc-1α, a target gene of miR-494, and tendency to increase in mitochondrial DNA were observed. Primary adipocytes derived from KO mice showed enhanced mitochondrial respiration driven by fatty acids as determined by extracellular flux analysis. Finally, KO mice showed an extended median lifespan 29% relative to WT mice (from 113.7 to 146.7 weeks). Aged KO mice (≥ 2 years old) showed reduced age-related changes, such as hair graying and low physical activity. In conclusion, deletion of miR-494 demonstrated increased adipocyte browning and survival advantage in mice. Although there are numerous studies that focused on promoting adipocyte browning, its long-term effect has not been well evaluated. Thus, our data showing the association among miR-494, adipocyte browning and longevity provide a novel insight into adipocyte biology and aging study. The gene sequence of miR-494 and its binding site to Pgc-1α are conserved from mice to humans; it is expected to be applied to human research. Disclosure L.Sugawara: None. K.Morino: Research Support; AstraZeneca, Astellas Pharma Inc., Sanwa Kagaku Kenkyusho, Ono Pharmaceutical Co., Ltd. H.Iwasaki: None. S.Ida: None. T.Yanagimachi: None. M.Lemecha: None. H.Maegawa: None. Y.Fujita: Research Support; Terumo Corporation, Boehringer Ingelheim Japan, Inc., Japan Society for the Promotion of Science, Mitsubishi Tanabe Pharma Corporation, Sumitomo Dainippon Pharma Co., Ltd. S.Kume: Research Support; Boehringer Ingelheim International GmbH, Japan Society for the Promotion of Science. Funding Grants-in-Aid for Scientific Research (22K08650)