Osteopontin, a major non-collagenous bone matrix protein, is strikingly upregulated in various tissues under certain pathologic conditions, including cancer. However, the mechanism of upregulation of the osteopontin gene in tumor cells remains unclear. Okadaic acid, a strong non-phorbol ester tumor promoter, is known to stimulate the expression of osteopontin. The aim of the present study was to understand the mechanism by which okadaic acid regulates osteopontin gene expression. Okadaic acid stimulated osteopontin mRNA expression in several cell lines within 3 h, and the increase in osteopontin mRNA was sustained for 24 h. New protein synthesis was required for the okadaic acid-elicited increase in osteopontin mRNA expression. A serial promoter deletion study showed that the okadaic acid-response element is located between positions -265 and -73, a sequence that includes the Runx2, Ets-1, and AP-1 binding sequences. Okadaic acid increased the mRNA expression of AP-1 components but not of Runx2 or Ets-1. Site-directed mutagenesis and electrophoretic mobility shift assays confirmed that protein binding of the AP-1 consensus sequence is necessary for the okadaic acid-mediated osteopontin gene upregulation. These results indicate that de novo induction of the oncoprotein AP-1 is required for okadaic acid-stimulated osteopontin gene upregulation.