Adipocyte renewal from preadipocytes has been shown to occur throughout life and to contribute to obesity, yet very little is known about the molecular circuits that control preadipocyte expansion. The soluble form of the preadipocyte factor (also known as pref-1) delta-like 1 homolog (DLK1(S)) is known to inhibit adipogenic differentiation; however, the impact of DLK1 isoforms on preadipocyte proliferation remains to be determined. We generated preadipocytes with different levels of DLK1 and examined differentially affected gene pathways, which were functionally tested in vitro and confirmed in vivo. Here, we demonstrate for the first time that only membrane-bound DLK1 (DLK1(M)) exhibits a substantial repression effect on preadipocyte proliferation. Thus, by independently manipulating DLK1 isoform levels, we established that DLK1(M) inhibits G1-to-S-phase cell cycle progression and thereby strongly inhibits preadipocyte proliferation in vitro. Adult DLK1-null mice exhibit higher fat amounts than wild-type controls, and our in vivo analysis demonstrates that this may be explained by a marked increase in preadipocyte replication. Together, these data imply a major dual inhibitory function of DLK1 on adipogenesis, which places DLK1 as a master regulator of preadipocyte homeostasis, suggesting that DLK1 manipulation may open new avenues in obesity treatment.