Adipogenesis is associated with the upregulation of the antioxidative enzyme manganese superoxide dismutase (MnSOD) suggesting a vital function of this enzyme in adipocyte maturation. In the current work, MnSOD was knocked-down with small-interference RNA in preadipocytes to study its role in adipocyte differentiation. In mature adipocytes differentiated from these cells, proteins characteristic for mature adipocytes, which are strongly induced in late adipogenesis like adiponectin and fatty acid-binding protein 4, are markedly reduced. Triglycerides begin to accumulate after about 6 days of the induction of adipogenesis, and are strongly diminished in cells with low MnSOD. Proteins upregulated early during differentiation, like fatty acid synthase and cytochrome C oxidase-4, are not altered. Cell viability, insulin-mediated phosphorylation of Akt, antioxidative capacity (AOC), superoxide levels, and heme oxygenase 1 with the latter being induced upon oxidative stress are not affected. L-Buthionine-(S,R)-sulfoximine (BSO) depletes glutathione and modestly lowers AOC of mature adipocytes. Addition of BSO to 3T3-L1 cells 3 days after the initiation of differentiation impairs triglyceride accumulation and expression of proteins induced in late adipogenesis. Of note, proteins that increased early during adipogenesis are also diminished, suggesting that BSO causes de-differentiation of these cells. Preadipocyte proliferation is not considerably affected by low MnSOD and BSO. These data suggest that glutathione and MnSOD are essential for adipogenesis.