Protease-activated receptors (PARs) play a clear role in the burst of inflammatory reactions and immune responses. However, for PAR-3, the most elusive member of the PAR family, the functional role is still largely unclear. It has been claimed that PAR-3 does not signal autonomously, although the wide expression of human PAR-3 indicates its important physiological roles. We demonstrate that in HEK-293 cells, stably transfected with human PAR-3, thrombin induced calcium signaling, IL-8 gene expression and IL-8 release. We confirmed this finding using human lung epithelial and human astrocytoma cells that express endogenous PAR-3. Moreover, thrombin exposure of HEK-293 cells resulted in ERK1/2 activation coinciding with IL-8 release. The effects of thrombin were not dependent on PAR-1 activation, as confirmed by PAR-1 gene silencing. Thus, we propose that PAR-3 is able to signal autonomously to induce IL-8 release mediated by ERK1/2 phosphorylation, which contributes actively to inflammatory responses.