Objective: The fat mass and obesity associated (FTO) gene is related to obesity, but the regulation of FTO expression in adipose tissue is not fully understood. We investigated FTO expression in paired subcutaneous and omental adipose tissues (SAT and OAT) from healthy women undergoing gynecological surgeries, and its relation with adiposity and insulin sensitivity. Design and Methods: FTO expression in SAT of type 2 diabetic patients treated or not with Rosiglitazone was also compared. Results: Both the mRNA and protein levels of FTO were higher in OAT from women than in SAT. Only OAT FTO protein levels negatively correlated with BMI and body fat mass, whereas SAT FTO mRNA levels were negatively correlated with subcutaneous fat deposition. In addition, SAT FTO mRNA and protein levels were increased in insulin resistant women (high HOMA) compared to insulin sensitive women (low HOMA), whereas OAT FTO expression was not different between these two subgroups. Interestingly, FTO mRNA levels were increased in SAT of type 2 diabetic patients, and treatment of diabetics with Rosiglitazone improved insulin sensitivity and reduced SAT FTO mRNA levels. Lastly, FTO expression was transiently increased in the early phase of 3T3-L1 cell differentiation, which coincides with the induction of PPARc2 expression. However, partial reduction of FTO did not impact PPARc2 expression and adipocyte differentiation. Conclusion: Therefore, FTO gene expression is higher in OAT than in SAT in lean to moderately obese women. OAT FTO expression is associated with adiposity, whereas SAT FTO expression is associated with insulin sensitivity. These associations are independent of an effect of FTO on adipocyte differentiation.