Multiple signaling pathways are involved in the tight regulation of Toll-like receptor (TLR) signaling, which is important for the tailoring of inflammatory response to pathogens in macrophages. It is widely accepted that TLR signaling can activate Notch pathway; however, whether full activation of Notch signaling can feedback modulate TLR signaling pathway so as to control inflammation response remains unclear. Here, we demonstrated that stimulation with TLR ligands up-regulated Notch1 and Notch2 expression in macrophages. The expression of Notch target genes including Hes1 and Hes5 was also induced in macrophages by LPS, suggesting that TLR4 signaling enhances the activation of Notch pathway. Importantly, overexpression of constituted active form of Notch1 (NICD1) and Notch2 (NICD2) suppressed production of TLR4-triggered proinflammatory cytokines such as TNF-α and IL-6 but promoted production of antiinflammatory cytokine IL-10, which is dependent on the PEST domain of NICD. In addition, NICD1 and NICD2 suppressed TLR-triggered ERK phosphorylation, which is indispensable for Notch-mediated inhibition of TLR4- triggered proinflammatory cytokine production. Furthermore, activation of Notch signaling inhibited NF-κB transcription activity by MyD88/TRAF6 and TRIF pathways, which was dependent on ERK activity. Therefore, our results showed that Notch signaling negatively regulates TLR-triggered inflammation responses, revealing a new mechanism for negative regulation of TLR signaling via Notch pathway.