The regulatory functions of glycosaminoglycan hyaluronan (HA) are suggested to be dependent on its molecular weight (MW). Proinflammatory and stimulatory effects are proposed mainly for the low MW HA. However, the complex response of blood phagocytes to HA of different MW is unclear. Herein, the effects of highly purified HA of precisely defined MW (52, 250, and 970 kDa) on human blood phagocytes were tested. All MW HA activated blood phagocytes, including the spontaneous production of ROS, degranulation, and the production of tumor necrosis factor alpha, with low MW HA 52 kDa having the highest potency and high MW HA 970 kDa having the lowest potency. Interestingly, HA inhibited ROS production stimulated by opsonized zymosan particles and, in contrast, potentiated starch-activated ROS production, mostly independent of MW. Data showed a significant effect of HA of different MW on blood phagocytes, including high MW HA.