Unwarranted overproduction of cytokines, such as interleukin (IL)-1β, can cause moderate to severe pathological complications, and thus elaborate mechanisms are needed to regulate its onset and termination. One such, well-known, mechanism is endotoxin tolerance, generally described as controlling lipopolysaccharide Toll-like receptor 4 (LPS-TLR4) signaling. Similarly, cytokine-induced tolerance plays an important role in regulating an overactive cytokine response. In this report, the capability of IL-1β to induce tolerance and cross-tolerance to various inflammatory ligands was investigated. IL-1β-stimulated THP-1 monocytes showed a gradual increase of microRNA (miR)-146a, reaching 15-fold expression by 24 h. miR-146a upregulation induced tolerance toward subsequent challenges of IL-1β, LPS, peptidoglycan, Pam and flagellin in THP-1 cells. The induction of tolerance was dependent on the IL-1β priming dose and associated increase of miR-146a expression. Moreover, IL-1β-treated THP-1 cells showed sustained miR-146a upregulation that repressed IRAK1 and TRAF6 adaptor molecules. Transfection of miR-146a alone mimicked IL-1β-induced tolerance in monocytes, while cells transfected with miR-146a inhibitor increased chemokine production. A comparable cytokine response regulated by miR-146a was also detected in lung epithelial A549 cells, purified human monocytes and mouse peritoneal macrophages. Thus, our studies showed that miR-146a was crucial for monocytic cell-based IL-1β tolerance and cross-tolerance, and thus opens the way for future research in the development of therapeutics for inflammatory diseases.