Background <p>The tumor microenvironment plays a critical role in cancer progression, with tumor-associated macrophages regulating immune responses. These macrophages can adopt a pro-inflammatory M1 phenotype that suppresses tumor growth or an anti-inflammatory M2 phenotype that promotes progression. Reprogramming macrophages toward the M1 phenotype is a therapeutic strategy. Previous studies showed that 4-Fluorophenylacetamide-acetyl coumarin (4-FPAC), a synthetic coumarin derivative, exhibits cytostatic activity in A549 lung carcinoma cells by modulating reactive oxygen species (ROS), nitric oxide synthase, and signaling pathways, including PI3K/AKT/NF-κB. This study evaluates the impact of 4-FPAC on macrophage polarization.</p> Hypothesis <p>We hypothesized that 4-FPAC promotes M1 macrophage polarization while suppressing M2 markers through modulation of signaling pathways, thus serving as an immunomodulatory agent.</p> Results <p>Treatment with 4-FPAC induced M1 polarization in THP1-derived macrophages, evident from morphological elongation, elevated ROS and NO production, and increased IL-12 levels. IL-10 levels and M2 markers (CD163, STAT3, AKT1) were downregulated, while M1 markers (CD80, STAT1, AKT2) were upregulated. Gene expression and western blot analyses revealed activation of P38 and NF-κB pathways and reduced phosphorylated AKT1 levels. In silico docking showed strong interactions of 4-FPAC with regulatory proteins like P38, NF-κB, and AKT1, suggesting pathway modulation.</p> Conclusion <p>4-FPAC facilitates M1 macrophage polarization and inhibits M2 signaling, demonstrating its potential as an immunomodulatory agent. Coupled with its cytostatic effects on A549 cells, these findings position 4-FPAC as a promising candidate for cancer therapy. Further in vivo studies are warranted to validate its therapeutic potential and explore applications in immunotherapy and inflammation-associated diseases.</p> Graphical Abstract <p>.</p> <p></p>