Toll-like receptors (TLRs) play an important role in host defense against invading pathogens. By initiating a signal transduction cascade, TLRs lead to the release of pro-inflammatory cytokines. However, the inappropriate activation of TLR signaling could result in inflammatory disorders or autoimmune diseases. Osteopontin (OPN) has been reported to be an inflammatory cytokine participating in cell-mediated immunity. However, the role of OPN in TLR-mediated immune responses is poorly understood. In the present study, OPN-deficient (OPN-/-) macrophages exhibited significantly higher levels of pro-inflammatory cytokines after stimulation with lipopolysaccharide (LPS). Our study also demonstrated that the intracellular OPN (iOPN) isoform acted as a negative regulator to inhibit LPS-induced inflammatory responses. Compared to WT macrophages, OPN-/- macrophages had lower Akt phosphorylation levels and higher GSK3β phosphorylation levels, which were downregulated by p-Akt. Moreover, as a down-stream target of Akt, 4EBP1 was hypo-phosphorylated in OPN-/- macrophages compared to 4EBP1 in WT macrophages. These findings reveal that iOPN can regulate GSK3β and 4EBP1 phosphorylation to inhibit TLR4-mediated inflammatory responses.