The adipocyte-specific secretory molecule adiponectin has found widespread acceptance as a systemic marker that effectively integrates a number of signals associated with metabolic dysfunction at the level of adipose tissue. The widely used aP2 promoter cassette, which is frequently chosen to achieve adipocyte-specific expression of transgenes, conveys transcription in cell types other than adipocytes, such as macrophages and cardiomyocytes. To improve our ability to drive transgene expression in a more adipocyte-specific way, we aimed to define the minimal promoter segment from the adiponectin genomic locus. We generated a series of transgenic animals in which the expression of reporter genes and Cre recombinase was driven by 2, 4.9, and 5.4 kb of adiponectin promoter sequences. We found that the 5.4-kb adiponectin promoter fragment is the most effective cassette conveying adipocyte-specific expression of target genes. We therefore define a novel promoter cassette that ensures adipocyte-specific expression of passenger genes and may be used in the generation of transgenic mouse models to study gene function in vivo.