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RNA splicing dysregulation and the involvement of specific splicing factors are emerging as common factors in both obesity and metabolic disorders. The study provides compelling evidence that the absence of the splicing factor SRSF1 in mature adipocytes results in whitening of brown adipocyte tissue (BAT) and impaired thermogenesis, along with the inhibition of white adipose tissue browning in mice. Combining single-nucleus RNA sequencing with transmission electron microscopy, it is observed that the transformation of BAT cell types is associated with dysfunctional mitochondria, and SRSF1 deficiency leads to degenerated and fragmented mitochondria within BAT. The results demonstrate that SRSF1 effectively binds to constitutive exon 6 of Ndufs3 pre-mRNA and promotes its inclusion. Conversely, the deficiency of SRSF1 results in impaired splicing of Ndufs3, leading to reduced levels of functional proteins that are essential for mitochondrial complex I assembly and activity. Consequently, this deficiency disrupts mitochondrial integrity, ultimately compromising the thermogenic capacity of BAT. These findings illuminate a novel role for SRSF1 in influencing mitochondrial function and BAT thermogenesis through its regulation of Ndufs3 splicing within BAT. Eliminating SRSF1 genetically triggers the whitening of brown adipocyte tissue (BAT) and compromises its thermogenic function, consequently causing metabolic disorders in mice. Combining single-nucleus RNA sequencing with transmission electron microscopy, the study highlights that the absence of SRSF1 disrupts mitochondrial homeostasis, leading to an overproduction of mitochondrial reactive oxygen species in BAT due to alterations in the splicing of Ndufs3. image