It is well-documented that nicotine, the main active ingredient in cigarettes, results in endothelial cell injury in numerous diseases. However, whether nicotine plays a crucial role in endothelial cell injury in diabetes and the exact molecular mechanism that mediates this process have not been fully elucidated. The current study aimed to investigate the effects of nicotine on endothelial cell injury in diabetes and the specific molecular mechanism by which it plays a role. Human umbilical vein endothelial cells (HUVECs) were incubated in HG/HF media and treated with nicotine, PYR-41 (a selective ubiquitin E1 inhibitor), Akt-overexpressing adenovirus, or TTC3 and MUL1 shRNA adenovirus. Cell viability was subsequently detected by the CCK8 assay, and apoptosis was examined by caspase-3 cleavage and activity analysis. Compared to the HG/HF incubated group, nicotine incubation significantly decreased cell survival and increased apoptosis. Moreover, nicotine induced Akt degradation via UPS, and Akt overexpression blocked nicotine-induced apoptosis in HUVECs cultured in HG/HF media. Furthermore, the TTC3 and MUL1 shRNA adenovirus dramatically decreased the Akt ubiquitination and apoptosis induced by nicotine. These results indicate that nicotine-induced Akt ubiquitination and degradation occurs through TTC3 and MUL1 and results in a dramatic increase in apoptosis in HUVECs cultured in HG/HF media.