Osteopontin (OPN) is expressed by a variety of immune cells and is critical for both innate and adaptive immune responses. The expression status of OPN might be tightly regulated to maintain immune homeostasis. However, the mechanisms by which OPN is negatively regulated in LPS-stimulated macrophages remain largely unknown. In this study, we showed that glycogen synthase kinase 3beta (GSK3beta) inhibitors - SB216763, LiCl and azakenpaullone - enhanced LPS-induced OPN expression in mouse peritoneal macrophages. GSK3beta knock-down had the similar effects. Furthermore, we found that GSK3beta inhibitors and GSK3beta knock-down both increased the activity of OPN promoter in LPS-stimulated macrophages. GSK3beta inhibitor-mediated enhancement of LPS-induced OPN promoter activity was abrogated in GSK3beta siRNA-treated macrophages. Therefore, we identified GSK3beta as a negative regulator of OPN expression and suggest GSK3beta as a potential therapeutic target for the intervention of diseases with uncontrolled OPN production.