Although microRNAs were shown to participate in innate immune responses, it is not completely understood how they regulate negative immunomodulatory events. IL-10 is an important anti-inflammatory mediator that prevents excessive inflammation and associated immunological pathologies. Although the regulation of IL-10 expression has been well studied at both the transcriptional and translational levels, it is less clear how microRNAs control IL-10 expression during inflammation. In this study, we found that miR-27a is downregulated in macrophages following stimulation through TLR2 and TLR4, but not TLR3. Upregulation of miR-27a enhanced the expression of proinflammatory cytokines in TLR2/4-activated macrophages. Conversely, knockdown of miR-27a diminished cytokine expression. Mechanistically, we found that miR-27a negatively regulates IL-10 expression; upregulation of miR-27a decreases, whereas downregulation of miR-27a increases, IL-10 expression in activated macrophages. Likely due to the decreased expression of IL-10, upregulation of miR-27a diminished IL-10-dependent STAT3 phosphorylation in TLR4-activated macrophages. Consistent with IL-10 being a potential mediator for the role of miR-27a in the immune response, blocking IL-10 abolished the enhancing effect of miR-27a on TLR4-activated inflammation. In conclusion, our study identified miR-27a downregulation as a negative-regulatory mechanism that prevents overly exuberant TLR2- and TLR4-driven inflammatory responses. Copyright © 2014 by The American Association of Immunologists, Inc.