Pyocyanin, an extracellular product of Pseudomonas aeruginosa (P. aeruginosa), is important in invasive pulmonary infection. Pseudomonas infections are characterized by a marked influx of polymorphonuclear cells (neutrophils). An increased release of interleukin-8 (IL-8), a potent neutrophil chemoattractant, in response to pyocyanin may contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage observed in Pseudomonas-associated lung diseases. In order to explore the pathogenesis of pyocyanin on macrophages, we treated phorbol-12-myristate-13-acetate (PMA)-differentiated U937 cells with pyocyanin in vitro and explored the expression of IL-8 using reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). In the present study, we also investigated the activation of protein kinase C (PKC) and nuclear factor (NF)-κB in PMA-differentiated U937 cells by western blotting and immunohistochemical methods. It was found that pyocyanin increased IL-8 release and mRNA expression in differentiated U937 cells in a concentration- and time-dependent manner. Calphostin C (Cal C), a protein kinase C (PKC) inhibitor, and pyrrolidine dithiocarbamate (PDTC) , an NF-κB inhibitor, blocked IL-8 expression in a concentration-dependent manner in pyocyanin-induced U937 cells. We concluded that pyocyanin promotes IL-8 secretion and mRNA expression in a concentration- and time-dependent manner and furthermore, that the PKC and NF-κΒ signaling pathways may be involved in the expression of IL-8 in pyocyanin-infected PMA-differentiated U937 cells.