Chronic inflammation is widely recognized as a contributing factor to various diseases, such as cancer, cardiovascular diseases, metabolic syndrome, and neurodegenerative diseases. Current anti-inflammatory medications, like non-steroidal anti-inflammatory drugs (NSAIDs), have limitations due to associated side effects and cost. Isorhamnetin, a significant bioactive flavonoid compound primarily found in the fruits of Hippophae rhamnoides L. and the leaves of Ginkgo biloba L., demonstrates diverse pharmacological effects, including its ability to regulate the immune response and potent antioxidant activity. Therefore, the objective of this study is to investigate the impact of Isorhamnetin on the inflammatory response in lipopolysaccharide-induced RAW 264.7 macrophages. Materials and methods: The effect of isorhamnetin on inflammatory mediators, cytokines, and reactive oxygen species was investigated using the RAW264.7 murine macrophage cell line. Cell viability was determined through the MTT assay, wherein RAW macrophages were exposed to LPS (1μg/ml) and treated with varying concentrations of isorhamnetin for 24 hours. Additionally, the effect of isorhamnetin on the production of nitric oxide and reactive oxygen species (ROS) was examined. Gene expression analysis was conducted using qPCR, and protein levels of pro-inflammatory cytokines were measured through enzyme-linked immunosorbent assay (ELISA). Results: The findings indicated that Isorhamnetin had a protective effect against LPS-induced toxicity, leading to enhanced cell viability at higher concentrations. Moreover, there was a substantial decrease in the production of nitric oxide and reactive oxygen species (ROS). Gene expression analysis demonstrated a significant downregulation of pro-inflammatory cytokines TNF-α, Cox-2, and iNOS in comparison to the LPS-only group following Isorhamnetin treatment. ELISA analysis also showed a marked reduction in protein levels of PGE2 and IL-6 when compared to the LPS-alone group. Conclusion: The results of the current investigation indicate that isorhamnetin exhibits a strong anti-inflammatory effect against with LPS-induced inflammation in RAW 264.7 macrophages.