Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) plays a crucial role in maintaining vascular homeostasis. As a hallmark of eNOS activation, phosphorylation of eNOS at Ser1177 induced by activated protein kinase B (PKB/Akt) is pivotal for NO production. The complete activation of Akt requires its phosphorylation of both Thr308 and Ser473. However, which site plays the main role in regulating phosphorylation of eNOS Ser1177 is still controversial. The purpose of the present study is to explore the specific regulatory mechanism of phosphorylated Akt in eNOS activation. Inhibition of Akt Thr308 phosphorylation by a specific inhibitor or by siRNA in vitro led to a decrease in eNOS phosphorylation at Ser1177 and to lower NO concentration in the cell culture medium of HUVECs. However, inhibiting p-Akt Ser473 had no effect on eNOS phosphorylation at Ser1177. Next, we administered mice with inhibitors to downregulate p-Akt Ser473 or Thr308 activity. Along with the inhibition of p-Akt Thr308, vascular p-eNOS Ser1177 protein was simultaneously downregulated in parallel with a decrease in plasma NO concentration. Additionally, we cultured HUVECs at various temperature conditions (37, 22, and 4 °C). The results showed that p-Akt Ser473 was gradually decreased in line with the reduction in temperature, accompanied by increased levels of p-Akt Thr308 and p-eNOS Ser1177. Taken together, our study indicates that the phosphorylation of Akt at Thr308, but not at Ser473, plays a more significant role in regulating p-eNOS Ser1177 levels under physiological conditions.