The bacterial N-3-oxo-dodecanoyl-L-homoserine lactone (C12) has critical roles in both inter-bacterial communication and inter-kingdom signaling. The ability of C12 to down-regulate production of the key pro-inflammatory cytokine tumor necrosis factor α (TNFα) in stimulated macrophages was suggested to contribute to the establishment of chronic infections by opportunistic Gram-negative bacteria, such as Pseudomonas aeruginosa. We show that in contrast to TNFα suppression, C12 amplifies production of the major anti-inflammatory cytokine interleukin-10 (IL-10) in lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophages as well as peritoneal macrophages. Furthermore, C12 increases IL-10 mRNA level and IL-10 promoter reporter activity in LPS-stimulated RAW264.7 macrophages, indicating that C12 modulates IL-10 expression at the transcriptional level. Finally, C12 substantially potentiated LPS-stimulated NFκB DNA-binding level, and prolonged p38 MAP kinase phosphorylation in the RAW264.7 macrophages, suggesting that increased transcriptional activity of NFκB and/or p38-activated transcription factors serves to up-regulate IL-10 production in macrophages exposed to both LPS and C12. These findings reveal another part of the complex array of host transitions through which opportunistic bacteria down-regulate immune responses in order to flourish and establish a chronic infection.