Objective To establish the method of suspension culture for stem cells from human pancreatic cancer cell line PANC1.Methods PANC1 cells were cultured in serum-free medium under floating-culture system.Tumor cell spheres were observed by optical microscope.Expression of CD133 and cell cycle were detected by flow cytometry.Cancer stem cells were induced to differentiate with 10%FBS,and expression of CK18,was evaluated with immunofluorescence microscope.Spheres cells were injected into the subcutaneous space of NOD/SCID rat and tumor formation was monitored weekly.Results PANC1 cells could form the stem cells spheres,and the rate of sphere formation was stable between 4%0 and 5%0 after 20 passages in vitro.The expression of CD133(5.91±0.7%)and proportion of G0/G1 phase cell(80.99±2.60%)was significantly increased in spheres cells compared with parental PANC1 cells(1.44±0.52%and 69.01±5.03%),and the difference was statistically significant(P＜0.05).When these spheres cells were cultured in media with serum,these cells gradually returned to the status of parental cells and expressed CK18,2×103 sphere cells injection could initiate tumor fornmtion in NOD/SCID rat.Conclusions Tumor spheres stem cellscould be generated under serum-free floating-culture system.The sphere cells possessed the capacities of self renew,difierentiation,and tumorigenic potential.