The positive effects of high glucose on the cellular productivity of nitric oxide (NO), and the mechanisms of the enhancement, were investigated. Macrophages were shifted from normal-glucose medium (5.5 mM) to high-glucose medium (25 mM) and immediately treated with lipopolysaccharide (LPS). Inducible nitric oxide synthase (iNOS) expression was expressed significantly more quickly, and NO production also increased. High-glucose conditions reduced cell viability at 48 h. Pretreatment with oxidized adenosine triphosphate (o-ATP), the selective purinergic receptor antagonist, strongly reduced LPS-induced iNOS expression, NO production and cell death in cells exposed to high levels of glucose. Apyrase, an ATP-hydrolyzing enzyme, also reduced the effects of high-glucose content. High-glucose content promoted the LPS-induced release of endogenous ATP from RAW 264.7 cells, as measured by luciferin-luciferase assay. In summary, the results revealed that purinergic receptor is important in responding to LPS challenge, increasing LPS-induced NO production and cell death under high-glucose conditions, and promoting the release of ATP from macrophages in high-glucose medium.