Endotoxin tolerance is a phenomenon characterized by a reduced capacity of monocytes and macrophages to respond to repeated stimulation with lipopolysaccharide (LPS) which has been suggested to represent a way of controlling the intensity and duration of innate immune response. During endotoxin tolerance, monocytes undergo functional re-programming primarily by epigenetic regulation. Recently, micro-RNA (miR)-146a has been demonstrated to be the major player of the negative regulation of the pro-inflammatory response, affecting TNF-α production. In this study, we have employed CyP, a cyanobacterial LPS antagonist acting on TLR4-MD2 complex, for priming human monocytes and evaluating their response to a subsequent challenge with E. coli LPS. Results show that CyP is able to induce cross-tolerance to E. coli LPS by inhibiting TNF-α production. The mechanism of action is mediated by a specific induction of miR-146a and reduction of IRAK1 and TRAF6 expressions in human monocytes by CyP priming. Up-regulation of miR-146a by CyP alone, affects subsequent cell response in term of TNF-α production even when monocytes are incubated with other TLR ligands, as lipoteichoic acid (LTA), thus confirming miR-146a as a critical player mediating TNF-α regulation during cross-tolerance with CyP.