N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils’ functional responses. However, though Ca2+ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca2+]?) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca2+]? in human neutrophils. We observed that NAC (1 μM ~ 1 mM) and cysteine (10 μM ~ 1 mM) increased [Ca2+]? in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucinephenylalanine (fMLP)-induced increase in [Ca2+]i in human neutrophils was observed. In Ca2+-free buffer, NAC- and cysteine-induced [Ca2+]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca2+]i in human neutrophils occur through Ca2+ influx. NAC- and cysteine-induced [Ca2+]? increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 μM) and ruthenium red (20 μM). In Na+-free HEPES, both NAC and cysteine induced a marked increase in [Ca2+]i in human neutrophils, arguing against the possibility that Na+-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca2+]? increasing activity. Our results show that NAC and cysteine induce [Ca2+]? increase through Ca2+ influx in human neutrophils via SKF96365- and ruthenium red-dependent way.