Human natural killer (NK) cell lines (YT and NK-92) and freshly isolated human NK cells were used to determine signal pathway (s) and their cytolysis-related molecules involved in IFNα-stimulated natural cytotoxicity. NK cells displayed apparently augmented cytotoxicity against target tumor cells (K562) and up-regulated gene expression of cytolytic effectors Fas-L and perforin in response to IFNα stimulation. Meanwhile, the tyrosine phosphorylation of STAT1 of NK cells was quickly induced, but other pathways including STAT3, STAT6, ERK1/2, p38 MAPK, JNK/SAPK, PI-3K, NF-κB were not or only weakly activated. Transient expression of dominant-negative form of STAT1 (DN STAT1) markedly inhibited STAT1 activation and then alleviated cytolysis activity of IFNα-treated YT cells, which was correlated to a markedly down-regulated expression of IRF-1, a key transcription factor necessary for cytotoxicity of IFNα/β-activated NK cells. The results indicate that STAT1 activation play a crucial role in IFNα signaling for cytolysis function of NK cells.