Aim To observe the effect of palmitic acid (PA) on inflammatory response of endothelial cell line EA.hy926 in vitro, and to detect the expression of related genes and explore their possible signaling pathways. Methods Endothelial cell line EA.hy926 was cultured in vitro and divided into albumin control group and PA intervention group. The effect of PA incubation on EA.hy926 cell viability was detected by modified MTT assay. mRNA of interleukin-1(IL-1), interleukin-6(IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α) and monocyte chemotactic protein 1(MCP-1) were detected by real-time fluorescence quantitative PCR. The levels of IL-6, IL-8 and TNF-α were detected by ELISA, and the levels of phosphorylated nuclear factor-κB (NF-κB) p65, NF-κB p65 and nuclear factor-kappa B inhibitor protein (IκBα) were detected by Western blot. Results Compared with the albumin control group, the mRNA levels of IL-1, IL-6 and IL-8 in PA (20 μmol/L, 50 μmol/L, 100 μmol/L) intervention group significantly increased (P<0.05). The mRNA levels of TNF-α and MCP-1 in PA (50 μmol/L, 100 μmol/L) intervention group significantly increased (P<0.05). The levels of IL-6, IL-8 and TNF-α in supernatant increased significantly (P<0.05), the level of IκBα protein decreased significantly (P<0.05), and the phosphorylation level of NF-kappa B p65 increased significantly (P<0.05). Conclusion PA intervention induces inflammatory response in EA.hy926 cells, which may be related to activation of NF-kappa B signaling pathway.