Icariside II, as a favonoid compound derived from epimedium, has been proved to involed in a variety of biological and pharmacological effects such as anti-inflammatory, anti-osteoporosis, anti-oxidation, anti-aging, and anti-cancer but its mechanism is unclear, especially in terms of its effect on post-transcriptional modification of endothelial nitric oxide synthase (eNOS). Phosphorylation of eNOS plays an important role in the synthesis of nitric oxide in endothelial cells, which is closely related to erectile dysfunction, atherosclerosis, Alzheimer's disease, and other diseases. Our study aims to investigate the effect and mechanism of Icariside II on the rapid phosphorylation of eNOS. In this study, human umbilical vein endothelial cells (HUVECs) were stimulated with Icariside II in the presence or absence of multiple inhibitors (1 µM), including LY294002 (PI3K-inhibitor), MK-2206 (AKT-inhibitor), Bisindolylmaleimide X (AMPK-inhibitor), H-89 (CaMKII-inhibitor), KN-62 (PKA-inhibitor), Dorsomorphin (PKC-inhibitor). The proliferation of HUVECs was assessed using cell counting kit-8 (CCK-8). The release of nitric oxide (NO) within HUVECs was detected via fluorescence probe (DAF-FM). Western blot was used to examine the effect of Icariside II on the expression of eNOS, phosphorylation of eNOS, and common signaling pathways proteins. In this study, Icariside II was found to promote the cell proliferation and rapid NO release in HUVECs. The phosphorylation of eNOS-Ser1177 was significantly increased after Icariside II stimulation and reached a peak at 10 min (p < 0.05). Meanwhile, the phosphorylation of eNOS-Thr495 was significantly decreased after 45 min of stimulation (p < 0.05). Following the intervention with multiple inhibitors, it was found that MK-2206 (AKT inhibitor), LY294002 (PI3K inhibitor), KN-62 (AMPK inhibitor), and Bisindolylmaleimide X (PKC inhibitor) could significantly inhibit the phosphorylation of eNOS-Ser1177 caused by Icariside II (p < 0.05), while MK-2206, LY294002, and Bisindolylmaleimide X reversed the alleviated phosphorylation of eNOS-Thr495. We concluded that Icariside can regulate rapid phosphorylation of eNOS- Ser1177 and eNOS-Thr495 via multiple signaling pathways, resulting in the up-regulation of eNOS and the increased release of NO.