GH influences adipocyte differentiation, but both stimulatory and inhibitory effects have been described. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are multipotent and are able to differentiate into adipocytes, among other cells. Canonical Wnt/β-catenin signaling activation impairs adipogenesis. The aim of the present study was to elucidate the role of GH on AT-MSC adipogenesis using cells isolated from male GH receptor knockout (GHRKO), bovine GH transgenic (bGH) mice, and wild-type littermate control (WT) mice. AT-MSCs from subcutaneous (sc), epididiymal (epi), and mesenteric (mes) AT depots were identified and isolated by flow cytometry (Pdgfrα+ Sca1+ Cd45- Ter119- cells). Their in vitro adipogenic differentiation capacity was determined by cell morphology and real-time RT-PCR. Using identical in vitro conditions, adipogenic differentiation of AT-MSCs was only achieved in the sc depot, and not in epi and mes depots. Notably, we observed an increased differentiation in cells isolated from sc-GHRKO and an impaired differentiation of sc-bGH cells as compared to sc-WT cells. Axin2, a marker of Wnt/β-catenin activation, was increased in mature sc-bGH adipocytes, which suggests that activation of this pathway may be responsible for the decreased adipogenesis. Thus, the present study demonstrates that (i) adipose tissue in mice has a well-defined population of Pdgfrα+ Sca1+ MSCs; (ii) the differentiation capacity of AT-MSCs varies from depot to depot regardless of GH genotype; (iii) the lack of GH action increases adipogenesis in the sc depot; and iv) activation of the Wnt/β-catenin pathway might mediate the GH effect on AT-MSCs. Taken together, the present results suggest that GH diminishes fat mass in part by altering adipogenesis of MSCs.