: Fractalkine (FKN) is a cytokine which plays an important role in atherosclerosis and other inflammatory diseases. Studies have shown that FKN induces integrin-independent leukocyte adhesion to primary endothelial cells under physiological flow conditions. Further, increased expression of FKN has been demonstrated in atherosclerotic lesions induced by low shear stress. However, the signal transduction mechanisms involved in low shear stress-induced FKN upregulation are not well characterized. In this study, EA.hy926 cells were subjected to varying intensity of fluid shear stress for different time durations. Further, mRNA and protein expressions of FKN were assessed by quantitative real-time PCR and Western blotting, respectively. Upregulation of FKN expression, which was induced via activation of mitogen-activated protein kinases signaling pathway under conditions of low shear stress, was studied both in the presence and absence of inhibitors. Low shear stress (∼4.58 dyne/cm) for more than 1 h promoted FKN expression and activated the extracellular signal-regulated kinase (ERK)1/2, p38, and Jun N-terminal kinase (JNK) mitogen-activated protein kinases signaling pathways by their phosphorylation. Inhibitors of ERK1/2, p38, and JNK pathways downregulated the FKN expression. In this study, fluid shear stress affected FKN expression in endothelial cells via activation of ERK1/2, p38, and JNK in a time-dependent manner. Our findings serve to advance the theoretical basis for prevention and treatment of atherosclerosis.