Abstract Ca 2+ plays a complex role in the differentiation of committed pre-adipocytes into mature, fat laden adipocytes. Stim1 is a single pass transmembrane protein that has an essential role in regulating the influx of Ca 2+ ions through specific plasma membrane store-operated Ca 2+ channels. Stim1 is a sensor of endoplasmic reticulum Ca 2+ store content and when these stores are depleted ER-localized Stim1 interacts with molecular components of store-operated Ca 2+ channels in the plasma membrane to activate these channels and induce Ca 2+ influx. To investigate the potential role of Stim1 in Ca 2+-mediated adipogenesis, we investigated the expression of Stim1 during adipocyte differentiation and the effects of altering Stim1 expression on the differentiation process. Western blotting revealed that Stim1 was expressed at low levels in 3T3-L1 pre-adipocytes and was upregulated 4 days following induction of differentiation. However, overexpression of Stim1 potently inhibited their ability to differentiate and accumulate lipid, and reduced the expression of C/EBP alpha and adiponectin. Stim1-mediated differentiation was shown to be dependent on store-operated Ca 2+ entry, which was increased upon overexpression of Stim1. Overexpression of Stim1 did not disrupt cell proliferation, mitotic clonal expansion or subsequent growth arrest. siRNA-mediated knockdown of endogenous Stim1 had the opposite effect, with increased 3T3-L1 differentiation and increased expression of C/EBP alpha and adiponectin. We thus demonstrate for the first time the presence of store-operated Ca 2+ entry in 3T3-L1 adipocytes, and that Stim1-mediated Ca 2+ entry negatively regulates adipocyte differentiation. We suggest that increased expression of Stim1 during 3T3-L1 differentiation may act, through its ability to modify the level of Ca 2+ influx through store-operated channels, to balance the level of differentiation in these cells in vitro.