Objective:To investigate the regulation mechanism of prestimulation of Lactobacillus paracacei (L.para) inhibited on LPS-induced inflammatory cytokine release. Methods:THP-1 cells were differentiated to macrophage-like cells by PMA stimulation. Cells were then pretreated by L.para (the experimental group),a lower dose LPS (10 ng/ml) and TLR2 agonist Pam3CSK4 respectively (the control group). After 24 hours pretreatment,THP-1 cells were further stimulated with a higher dose LPS (1 -滋g/ml). The influence of this prestimulation on the expression of TLR signaling pathway negative regulators,membrane expression and mRNA levels in TLR4 and CD14 and IL-1β and TNF-α levels in a higher dose LPS-induced inflammatory cytokines were respectively detected. mRNA levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR). Membrane expressions of TLR4 and CD14 after pretreatment were confirmed by flow cytometry. Results:LPS-induced IL-1β and TNF-α levels were significantly reduced by L.para pretreatment;In addition,the mRNA level of TLR signaling pathway negative regulators A20,SOCS1,SOCS3 and IRAK3 were upregulated while membrane expression of TLR4 and CD14 unaltered. Prestimulation-reduced negative regulators expression levels along with the release of LPS-reduced inflammatory cytokines were weaken by a IRAK1/4 inhibitor. Conclusion:L.para prestimulation could enhance negative regulators of TLR signaling A20,SOC1,SOCS3 and IRAK3 expression thus inhibits inflammatory response of macrophage after LPS stimulation.