MicroRNAs (miRNAs) are endogenous small non-coding RNAs which modulate gene expression at the post-transcriptional level by either translational inhibition or mRNA degradation. MicroRNAs play important roles in both innate and adaptive immune response, including TLR-triggered immune response. In this study, we found that the expression of miR-709 was up-regulated in primary macrophage and RAW264.7 cells during the stimulation of LPS. Overexpression of miR-709 in RAW264.7 cells led to reduced production and gene expression of inflammatory cytokines (IL-6, TNF-α, IL-1β) during activation by LPS, whereas knockdown of miR-709 had completely opposite effects. We used bioinformatics and experimental techniques to demonstrate that GSK-3β is a direct target of miR-709. miR-709 mimics decreased GSK-3β protein but not mRNA level. We also found that miR-709 regulated the LPS-induced inflammatory response by targeting GSK-3β and elevating β-catenin. In conclusion, our data revealed a novel role for miR-709 in regulation of inflammatory response by targeting GSK-3β.