Osteopontin (OPN), expressed by various immune cells, modulates both innate and adaptive immune responses. Different immune cells have shown differential expression of the two isoforms of OPN: secreted form of OPN (sOPN) and intracellular form of OPN (iOPN). However, the molecular mechanisms that control opn gene expression and the OPN isoforms produced by immune cells remain largely unknown. In this study, we demonstrate that OPN mRNA and protein expression are significantly up-regulated upon stimulation with TLR agonists in macrophages. Interestingly, we find that macrophages constitutively express the secreted form of OPN (sOPN), while the intracellular form of OPN (iOPN) is induced following the stimulation with TLR agonists. Phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and c-Jun NH(2)-terminal kinase (JNK) that are activated by LPS stimulation were shown to upregulate OPN expression. In addition, chromatin immunoprecipitation (CHIP) assays showed that AP-1 binds to the proximal AP-1 site in the OPN promoter from LPS-stimulated macrophages. Mutation of the AP-1 site in OPN promoter completely ablates LPS-induced OPN promoter activation. Knockdown of c-Jun and c-Fos expression by small interfering RNA (siRNA) significantly decreases LPS-induced OPN expression. Stable cell lines with iOPN overexpression and knockdown showed that TLR-induced iOPN expression is a negative regulator for interferon-beta (IFN-beta) production. Our findings provide new insight into the transcriptional regulation of opn gene and further clarify the isoforms and functions of OPN produced by macrophages.