Aims Oxidative stress of adipose tissue (AT) emerges as an instigator of metabolic dysfunctions in obese insulin-resistant patients. Here we test the hypothesis whether free radical-derived cholesterol oxidation products (i.e., oxysterols) are: 1) generated in the dysfunctional “diabetic” AT, 2) secreted in AT interstitial fluid, 3) and active as modulators of AT stem cell (ASC) adipogenic differentiation. 7-ketocholesterol (7k-C) and 7β-hydroxycholesterol (7β OH-C) were assessed in AT microdialysis samples of healthy subjects as well as in subcutaneous abdominal AT of type 2 diabetic obese patients. Adipogenic differentiation and “canonical” Wnt and MAPK signaling were investigated in primary cultures of human ASCs treated with 7k-C or with the autoxidation-type II oxysterol 5,6-secosterol (5,6-S) for comparison. Results 7k-C and 7β OH-C were detected in human AT interstitial fluid (0.27±0.03 and 0.91±0.13 μM, respectively). When compared with non-diabetic individuals, diabetic obese patients showed increased levels of 7k-C and 7β OH-C, as well as of 4-HNE-protein adducts, in subcutaneous AT. Challenging ASCs with 7k-C at levels resembling those found in vivo (1 μM), resulted in a lowered adipogenic differentiation by the transient increase of cellular ROS and sequential activation of Wnt/β-catenin, ERK1/2, p38-MAPK and JNK signaling. Signaling data differentiated the anti-adipogenic response of 7k-C and 5,6-S. Innovation and Conclusion Free radical-derived oxysterols were identified in the human AT secretome and may act as novel adipokines with regulatory effects on ASC differentiation. Pathogenic effects of these molecules and particularly of 7k-C in the impaired de novo adipogenesis of insulin-resistant patients is worth of further investigation.