Adipose tissue has long been considered to be a simple tissue that contains adipocytes. Because of this, the isolation and characterization of microvascular endothelical cells, which are also present in adipose tissue, have been neglected, even though they are components of a capillary network that surrounds each individual adipocyte. Here we report on a protocol for producing highly purified murine microvascular endothelial cells (MECs) from diverse sites of murine adipose tissues including inguinal and epididymal adipose tissues. The method is based on a combination of negative and positive immunomagnetic selection. The protocol involves the preparation of a single cell suspension (digestion, filtration and density gradient centrifugation), immunomagnetic enrichment of the CD45(-) cell population and the purification of the MECs by a combination of common specific markers CD31, CD102 and isolectin B4. The isolated MECs can be successively cultured for 10 to 12 passages without any detectable changes in morphology and phenotype. Therefore, the method described herein represents a protocol for the isolation and long-term maintenance of highly pure mouse MECs in high yields from adipose tissues.