Heparin is primarily used as an anticoagulant but has many biological functions as well. It binds with high affinity to a range of cytokines including interferon-γ (IFN-γ) and members of chemokine superfamily. IFN-γ is a proinflammatory cytokine that plays a pivotal role in immune and inflammatory responses; and in endothelial cells, it regulates the expression of fractalkine/CX3CL1 that is a potent agonist for the chemotaxis and adhesion of monocytes and lymphocytes. We have investigated the effect of heparin on the fractalkine expression in human umbilical vein endothelial cells (HUVEC) in culture. HUVEC were treated with ∼100 μg/mL heparin and the expression of the IFN-γ-induced fractalkine mRNA and protein were measured by reverse transcription-PCR and western blotting. The IFN-γ-induced expressions of fractalkine mRNA and protein were inhibited by heparin in a concentration-dependent manner. Heparin also inhibited adhesion of mononuclear cells (MNC) to HUVEC monolayers stimulated with IFN-γ, but it did not inhibit the MNC adhesion to the monolayers stimulated with interleukin-1β. Electrophoretic analysis demonstrated direct binding of heparin to IFN-γ and heparin was found to partially block the binding of IFN-γ to IFN-γ receptor (IFN-γ R). Heparin may play a regulatory role in inflammatory and immune responses by modulating the interaction between leukocytes and the vascular endothelium.