Summary Stromal cells are the most abundant cell population present in decidual tissue; they are involved in key processes during embryo implantation, fetal nutrition and the pregnancy maintenance. Described procedures for stromal cells isolation require the use of many monoclonal antibodies due to contamination with another cell types in the decidua; besides, some markers of stromal cells show variability during the days of gestation. In this study, we standardized a procedure for isolation by enzymatic digestion, density gradient and adherence to plastic. Murine stromal cells were characterized by exclusion of markers that are expressed in macrophages (F4-80), epithelial cells and trophoblast (cytokeratin-7), yielding a 98% of negative cells for these markers that correspond to stromal cells. This isolation procedure permits to obtain stromal cells with less expensive and high efficiency methods that provide a useful cellular model to study the physiology of gestation in different species.