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Background and Objectives: Macrophages play an important role in tumor growth (M2 macrophage) or suppression (M1 macrophage). Auraptene, a prenyloxycoumarin compound extracted from Citrus plants, has anti-cancer and anti-inflammatory properties. The purpose of this study was to look into the effect of auraptene on macrophage polarization and the tumor microenvironment when a human monocyte cell line (THP-1) was co-cultured with human colorectal adenocarcinoma (HT-29). Methods: The toxicity of auraptene on THP-1 and HT-29 cells was determined by the MTT method. Using flow cytometry, the effect of auraptene on macrophage polarization was studied through THP-1 as a macrophage source. The effect of auraptene on the macrophage population was also studied in THP-1 co-cultured with HT-29. Furthermore, macrophage function was assessed by measuring IL-10 and IL-12 concentrations using the ELISA method, nitric oxide (NO) concentrations using the Griess method, and HT-29 apoptosis by flow cytometry. Results: The M1/M2 ratio of THP-1 exposed to auraptene increased significantly in both naive THP-1 and THP-1 co-cultured with HT-29. Auraptene significantly reduced tumor-protective IL-10 secretion in M1 (p=0.0032) and M2 (p=0.0011). Auraptene increased anti-tumor IL-12 in M2 significantly (p=0.0011). It increased M1 NO production (p=0.0236) while decreasing M2 NO production (p=0.0001). Auraptene also increased HT-29 apoptosis in M0 and M1 co-cultures (p