Objective This study explores the mechanism of cyclooxygenase-2 (COX-2) upregulation in oral cancers asso- ciated with macrophage by using molecular biology techniques and primary culture of murine macrophage.Methods Murine macrophage was induced by macrophage colony-stimulating factor (M-CSF) and Cal27 conditional medium (CM). Purity of the macrophage was detected through CD68 immunofluorescence staining. Inhibitors of spleen tyrosine kinase (Syk) and nuclear factor κB (NFκB) were used to inhibit these pathways. In addition, real-time polymerase chain reaction and Western blot analysis were used to detect alterations in COX-2 and pathway-related proteins.Results All of the induced cells specifically expressed CD68. Cal27 CM could significantly induce COX-2 expression (PConclusion Syk-NFκB is responsible for COX-2 overexpression in oral cancer associated with macrophages. Targeting this pathway is possibly a new approach to control oral cancer-related pain.