The increase of extracellular glycine concentration prevents or mitigates a variety of pathological dysfunctional inflammatory responses. To eliminate the systemic effects of glycine as the reduction in the release of cytokines, this study was performed in isolated human neutrophils. The increase of the intracellular calcium concentration ([Ca2+](i)) and reactive oxygen species (ROS) release in cells incubated with glycine (0.1 to 10 mM) and stimulated with fMLP or PMA were compared with glycine-free controls. Glycine inhibited ROS production but increased [Ca2+](i) signal produced by fMLP. The inhibition of ROS production was observed even when glycine was added after the ROS release had reached maximal rate. The inhibitory effect was insensitive to strychnine and also obtained when PMA was used as stimulant. This study demonstrated that glycine impaired the activation of oxidative burst independently of glycine-gated chloride channel, presumably at the membrane level.