Developmental genes are important regulators of fat distribution and adipose tissue (AT) function. In humans, the expression of homeobox c9 (HOXC9) is significantly higher in subcutaneous compared to omental AT and correlates with body fat mass. To gain more mechanistic insights into the role of Hoxc9in AT, we generated Fabp4-Cre-mediated Hoxc9 knockout mice (ATHoxc9(-/-)). Male and female AT Hoxc9(-/-)mice were studied together with littermate controls both under chow diet (CD) and high-fat diet (HFD) conditions. Under HFD, only male ATHoxc9(-/-)mice gained less body weight and exhibited improved glucose tolerance. In both male and female mice, body weight, as well as the parameters of glucose metabolism and AT function were not significantly different between ATHoxc9(-/-) and littermate control CD fed mice. We found that crossing Hoxc9 floxed mice with Fabp4-Cre mice did not produce a biologically relevant ablation of Hoxc9in AT. However, we hypothesized that even subtle reductions of the generally low AT Hoxc9 expression may cause the leaner and metabolically healthier phenotype of male HFD-challenged AT Hoxc9(-/-)mice. Different models of in vitro adipogenesis revealed that Hoxc9 expression precedes the expression of Fabp4, suggesting that ablation of Hoxc9 expression in AT needs to be achieved by targeting earlier stages of AT development.