Quorum-sensing systems is critical regulator of the expression of virulence factors of various organisms, including Pseudomonas aeruginosa. Las and Rhl are two major quorum-sensing components, and they are regulated by their corresponding autoinducers, N-3-oxododecanoyl homoserine lactone (3-oxo-C₁₂-HSL) and N-butyryl-L-homoserine lactone (C₄-HSL). Recent progress has demonstrated the potential of quorum-sensing molecules, especially 3-oxo-C₁₂-HSL, for modulation of the host immune system. Here we show the specific ability of 3-oxo-C₁₂-HSL to induce apoptosis in mice-derived macrophages (RAW264.7) and reduce phagocytosis of the cell. When RAW264.7 cells were incubated with synthetic 3-oxo-C₁₂-HSL, the significant loss of viability was observed in a concentration (6.25 to 100 μmol/L) and incubation time (2 to 24 h) dependent manner. The cytotoxic activity of 3-oxo-C₁₂-HSL was also observed in RAW264.7 cells. The cells treated with 3-oxo-C₁₂-HSL revealed morphological alterations indicative of apoptosis. Acceleration of apoptosis in 3-oxo-C₁₂-HSL-treated cells was confirmed by multiple criteria (caspases 3, 8 and 9, mitochondrial depolarization, phosphatidylserine expression). Phagocytosis of neutral red assay demonstrated that higher concentration of 3-oxo-C₁₂-HSL significantly reduced phagocytosis of RAW264.7 cells (P < 0.05). High concentration of 3-oxo-C₁₂-HSL also obviously lowered RAW264.7 cells for gobbling up of P. aeruginosa capability (P < 0.001). These data suggest that 3-oxo-C₁₂-HSL specifically promotes induction of apoptosis and reduces the phagocytosis of RAW264.7 cells, which may be associated with 3-oxo-C₁₂-HSL-induced cytotoxicity in RAW264.7 cells. Our data suggest that the quorum-sensing signal molecule 3-oxo-C₁₂-HSL has critical roles in the pathogenesis of P. aeruginosa infection, not only in the induction of bacterial virulence factors, but also in the modulation of host responses.