Abstract The imprinted gene Dlk1, encodes a transmembrane protein, which may act as a negative regulator of Notch, and a soluble form of DLK1 (sDLK1) is generated by ectodomain shedding by ADAM17/TACE. DLK1 participates in mouse adipose tissue development by inhibiting adipocyte differentiation, and is generally referred to as “the” preadipocyte marker. The DLK1 gene itself is situated in one of the largest known miRNA clusters, yet, no reports have been made on miRNAs targeting Dlk1. By examinating preadipocytes in vitro using flowcytometry, ELISA, coulter cell counting, immunocytochemistry, qRT-PCR, and LNA based pre/anti-miR transfections, we here report that DLK1 levels in preadipocytes may be regulated by miR-15a, a microRNA previously implicated in cell cycle arrest and cancer. At present, accurate normalization of qRT-PCR miRNA data by multiple stably expressed miRs showed that miR-15a peaks in confluent layers of proliferating preadipocytes, the same stage where membrane- and shedded DLK1 are found at maximum levels during in vitro adipogenesis. Inhibition of miR-15a in preadipocytes by anti-miR 15a transfection resulted in significantly (P=0.01) higher (+6±3.1% geometric mean-fluorescence intensity) levels of membrane bound DLK1, but lower (-27.4 ±0.85%; P=0.03) amounts of shedded sDLK1 in the medium compared with scramble control transfected cells. In agreement with previous reports, we found that anti-miR 15a treatment affected cell numbers, where we observed a 20.4±5.6% cell number increase (P=0.005 vs. scramble) that was completely reverted by exogenous added sDLK1. Moreover, we found a dose-dependent knock-down of membrane bound DLK1 by pre-mir 15a treatment further supporting that Dlk1 is affected by miR-15a, and luciferase assays suggested that this interaction could be a consequence of Dlk1 being a direct target for miR-15a. Our data thus imply that miR-15a mediated cell cycle arrest may arise as a result of miR-15a regulating the levels of membrane versus shedded DLK1, and we hypothesize that this scenario not only has an impact on preadipocyte proliferation/differentiation, but also remains an issue in other cell lineages as well as cancer diseases where miR-15a deletions and increased levels of DLK1 have been described independently in numerous reports.