E-selectin expression by endothelial cells (ECs) is crucial for leukocyte recruitment during the inflammatory response. Macrophage accumulation and serum E-selectin elevation are features of type 2 diabetes mellitus. However, the interactions between macrophages and ECs in regulating vascular endothelial function are not clearly understood. We investigated the mechanisms underlying the modulation of EC E-selectin expression by high glucose (HG)-treated macrophages. Macrophage-conditioned media (MCM) were prepared from HG-treated macrophages. EC stimulation with HG-MCM induced increases the expression and secretion of E-selectin. By using specific inhibitors and small interfering RNAs, we demonstrate that the activation of the JNK and p38 MAPK pathways are critical for HG-MCM-induced E-selectin expression. Transcription factor ELISA and chromatin immunoprecipitation assays further showed that HG-MCM increases the NF-κB- and AP-1 DNA-binding activities in ECs. The inhibition of NF-κB and AP-1 activation by specific siRNAs blocks the HG-MCM-induced E-selectin promoter activity and expression. Protein arrays and blocking assays using neutralizing antibodies demonstrated that macrophage inflammatory protein 1α and 1β in HG-MCM are major mediators for the induction of EC E-selectin expression. These data support the hypothesis that E-selectin up-regulation stimulated by macrophages may play an active role in atherogenesis in the HG condition and suggest a new mechanism by which arterial disease is accelerated in diabetes.