The present study aimed to develop a quick and efficient method for purification of newborn endothelial cells from tumor tissues. Fresh tissues were separated from C57BL/6 mice bearing tumors derived from mouse lung cancer Lewis cells, fully minced and divided into two parts. One part was subjected to collagenase type I digestion with a vortex to form a single-cell suspension, while another part was digested but without a vortex. Then, the CD105+ cells were isolated using anti-CD105 antibody-coated Dynabeads. The isolated CD105+ cells were grown in culture medium and examined for the surface expression of CD105 by a fluorescence-activated cell sorter (FACS). The uptake of acetylated LDL and the ability to maintain capillary tube-like structure formation in the CD105+ cells were also examined by Dil-Ac-LDL uptake assay and tube formation assay. The expression of tumor newborn endothelial cells (CD105+) was tested in Lewis xenografts by immunohistochemistry. The number of cells which were obtained by the digestion process with a vortex was 5.70±0.23x10(4) much higher than the number without a vortex (0.32±0.04x10(4)) (P<0.01). The purity of CD105+ cell digestion with a vortex was significantly higher than that without a vortex. Dil-Ac-LDL uptake assay and tube formation assay confirmed that the CD105+ cells digested with a vortex exhibited typical functions of endothelial cells. In conclusion, the CD105+ cells isolated by the new method had high purity and displayed features of vascular endothelial cells. The modified method provides CD105+ cells with superior conditions for mechanistic research on the development of vessel-based disease.