Factor XIII-A (FXIII-A) transglutaminase (TG) was recently identified as a potential causative obesity gene in human white adipose tissue (WAT). Here, we have examined the role of TG activity and the role of protein crosslinking in adipogenesis. Mouse WAT and preadipocytes showed abundant TG activity arising from FXIII-A. FXIII-A was localized to the cell surface and acted as a negative regulator of adipogenesis by promoting assembly of fibronectin (FN) from plasma into preadipocyte extracellular matrix. This modulated cytoskeletal dynamics and maintained the preadipocyte state. FXIII-A–assembled plasma FN (pFN) matrix promoted preadipocyte proliferation and potentiated the proproliferative effects of insulin (INS) while suppressing the prodifferentiating INS signaling. FXIII-A–deficient mouse embryonic fibroblasts showed increased lipid accumulation and decreased proliferation as well as decreased pFN assembly into extracellular matrix. Thus, FXIII-A serves as a preadipocyte-bound proliferation/differentiation switch that mediates effects of hepatocyte-produced circulating pFN.