Abstract Objectives Lipedema, a poorly understood chronic disease of adipose hyper-deposition, is often mistaken for obesity and causes significant impairment to mobility and quality-of-life. To identify molecular mechanisms underpinning lipedema, we employed comprehensive omics-based comparative analyses of whole tissue, adipocyte precursors (adipose-derived stem cells (ADSCs)), and adipocytes from patients with or without lipedema. Methods We compared whole-tissues, ADSCs, and adipocytes from body mass index–matched lipedema (n = 14) and unaffected (n = 10) patients using comprehensive global lipidomic and metabolomic analyses, transcriptional profiling, and functional assays. Results Transcriptional profiling revealed >4400 significant differences in lipedema tissue, with altered levels of mRNAs involved in critical signaling and cell function-regulating pathways (e.g., lipid metabolism and cell-cycle/proliferation). Functional assays showed accelerated ADSC proliferation and differentiation in lipedema. Profiling lipedema adipocytes revealed >900 changes in lipid composition and >600 differentially altered metabolites. Transcriptional profiling of lipedema ADSCs and non-lipedema ADSCs revealed significant differential expression of >3400 genes including some involved in extracellular matrix and cell-cycle/proliferation signaling pathways. One upregulated gene in lipedema ADSCs, Bub1, encodes a cell-cycle regulator, central to the kinetochore complex, which regulates several histone proteins involved in cell proliferation. Downstream signaling analysis of lipedema ADSCs demonstrated enhanced activation of histone H2A, a key cell proliferation driver and Bub1 target. Critically, hyperproliferation exhibited by lipedema ADSCs was inhibited by the small molecule Bub1 inhibitor 2OH-BNPP1 and by CRISPR/Cas9-mediated Bub1 gene depletion. Conclusion We found significant differences in gene expression, and lipid and metabolite profiles, in tissue, ADSCs, and adipocytes from lipedema patients compared to non-affected controls. Functional assays demonstrated that dysregulated Bub1 signaling drives increased proliferation of lipedema ADSCs, suggesting a potential mechanism for enhanced adipogenesis in lipedema. Importantly, our characterization of signaling networks driving lipedema identifies potential molecular targets, including Bub1, for novel lipedema therapeutics.