White adipocytes play important roles in many physiological processes, including energy storage, endocrine signaling, and inflammatory responses. Understanding the molecular mechanisms of adipocyte formation (adipogenesis) provides insights into therapeutic approaches against obesity and its related diseases. Many transcriptional factors and epigenetic enzymes are known to regulate adipogenesis; however, whether histone variants play a role in this process is unknown. Here we found that macroH2A1.1 (mH2A1.1), a variant of histone H2A, was upregulated during adipocyte differentiation in 3T3-L1 cells and in the white adipose tissue of obese mice. Ablation of mH2A1.1 activated Wnt/β-catenin signaling pathway, while overexpression of mH2A1.1 showed opposite effects. We further found that mH2A1.1 regulated Wnt/β-catenin signaling pathway by cooperating with EZH2, a histone H3K27 methyltransferase, thus led to accumulation of H3K27me2 and H3K27me3 on the promoters of Wnt genes. Mutations in the macro-domain, mH2A1.1G224E, and mH2A1.1G314E, not only impaired adipogenesis, but also impaired the binding ability of mH2A1.1 to EZH2 and the enrichments of H3K27me2 and H3K27me3 on the promoters of Wnt genes. Together, our study reveals a novel regulatory role of mH2A1.1 in adipogenesis and obesity, which provides new insights in white fat development.