Endocan, a secretary proteoglycan, known to induce vascular inflammation. Nitric oxide (NO) produced by endothelial cells is an important signaling molecule in maintaining the vascular homeostasis. However, the precise effect of endocan in regulating NO pathway is not known. The present study explores the effect of endocan on eNOS-iNOS-NO and ROS production in cultured endothelial cells. Results showed that recombinant endocan treatment in HUVEC could increase NO and nitrite levels. However, pharmacological inhibition of iNOS using 1400W significantly decreased these effects. Furthermore, protein expression analysis showed that endocan could inhibit AKT/eNOS pathway and activate NF-κB/iNOS pathway. The production of superoxide, hydrogen peroxide, peroxynitrite and total ROS were also significantly increased with endocan treatment supported by decreased activity of superoxide dismutase and catalase. Moreover, selective inhibition of NOX reduced the ROS formation. In addition, mRNA expression analysis demonstrated that endocan can upregulate the expression of NOX1, NOX2 and NOX4. These findings suggest that endocan alters the NO production and their by enhances oxidative stress in endothelial cells. Thus, inhibition of endocan-NO signaling could be a one of the strategy to reduce oxidative stress in vascular disease.