Objective To explore how hyaluronic acid (HA) in extracellular matrix regulates the adhesion of CD44+ tumor cells. Methods MDA-MB-231 cells or HL60 cells were perfused in a parallel plate chamber. The movement of cells over immobilized HA was observed and analyzed to obtain the characteristics of adhesion and rolling. Results The adhesion number of MDA-MB-231 cells on HA substrate was positively regulated by HA concentration, but not by HA molecular weight; compared with physical adsorbed HA, immobilized HA by avidin-biotin could significantly improve the cell adhesion ratio. With the increase of shear stress in the range of 0.03 ~ 0.05 Pa, the rolling velocity of cells raised and the adhesion ratio decreased, but the tether lifetime of cells was not affected. In the same flow field, compared with MDA-MB-231 cells, HL60 cells with low expression of CD44 rolled quicker on immobilized HA with shorter tether lifetime and much lower adhesion ratio (< 1.5%). Conclusions: Fluid shear stress might mediate the rolling velocity of MDA-MB-231 cells by regulating the CD44-HA association rate rather than their dissociation rate. The interaction between CD44 and HA is involved in the initial adhesion of HL60 cells, but it does not play a major role. This study would be helpful for the design of anti-tumor drugs.