Objective To detect the expression level of miRNA-575 by pancreatic cancer stem cells.Methods Pancreatic cancer cell lines were cultured with serum-free medium.The cancer stem cells were obtained and their characteristics were observed with an invasion assay,a xenograft experiment,and flow cytometry analysis.Moreover,qRT-PCR was used to detect the expression of miR-575.Results A small subpopulation of cells in both ASPC-1 and PANC-1 cells survived and formed spheres in the absence of serum.The mean number of migrated cells of PANC-1 and ASPC-1 spheres was (147.3±18.6) cells and (113.2±12.9) cells.Therefore,the spheres have higher invasive ability compared with those of parental cells.As few as 5 × 105 cells in PANC-1 and ASPC-1 spheres could initiate a xenograft tumor in NOD/SCID mice,which suggested the tumorigenicity of the spheres was 100 times those of parental cell lines.The percentages of CD24+CD44+ in spheres were 0.38％-0.43％ and 4.91％-5.21％,which showed increased expression compared with ASPC-1 and PANC-1 cell lines (P＜0.05).The expression of miR-575 in spheres was 3.71 and 3.83 times its expression in cell lines ASPC-1 and PANC-1 respectively (P＜0.05).Conclusions Pancreatic cancer cell spheres may be a source for collecting cancer stem cells in a serum-free medium culture,and MiR 575 may play an important role in regulating the biological characteristics of pancreatic cancer stem cells.