The aim of this study was to clarify the role of diacylglycerol kinase (DGK)γ in vascular endothelial cells. The mRNA and protein expression of DGKγ and inducible nitric oxide synthase (iNOS) in rat aortic endothelial cells (RAECs) were investigated using RT-PCR, immunocytochemical, and immunoblot analyses. In RAECs, immunoreactivity of DGKγ was detected in the cytoplasm as a tubular or reticular structure. DGKγ immunoreactivity colocalized with those for GM130 and Golgin 97 but not with that for protein disulfide isomerase (PDI). In the presence of brefeldin A, DGKγ immunoreactivity was markedly decreased and displayed an aggregation-like pattern. After treatment of RAECs with nocodazole, DGKγ immunoreactivity was detected in Golgi stacks, which were severely segmented and appeared in vesicular shape. Stimulation with IL-1β increased mRNA expression of DGKγ, which was strongly attenuated by SB203580, a p38 MAPK inhibitor. IL-1β also induced expression of iNOS, which was observed as a tubular structure, and this distribution coincided with DGKγ immunoreactivity. Brefeldin A reduced both iNOS immunoreactivity and DGKγ immunoreactivity. iNOS expression was impaired by DGK inhibitors, R59022 and R59949. These results suggest that DGKγ is upregulated by IL-1β through the p38 MAPK pathway and may be involved in protein trafficking of iNOS in vascular endothelial cells.