The present study aimed to explore the effects of shikonin (SKN) on the damage of human venous endothelial cells (HUVECs) induced by ox-LDL and the underlying molecular mechanism. The HUVECs were randomly divided into six groups: control, ox-LDL, SKN + ox-LDL, SKN + ox-LDL + compound C, SKN + ox-LDL + si-Nrf2, and SKN + ox-LDL + si-HO-1. The MTT method was used to detect cell viability, flow cytometry was used to detect cell apoptosis and reactive oxygen species (ROS) levels, and Western blot was used to detect protein levels. Compared to the control group, the cell viability of the ox-LDL group decreased, the apoptosis rate increased, the level of cleaved caspase-3 was upregulated, and the level of Bcl-2 protein was downregulated. The level of TNF-α, IL-1β, IL-6, vascular cell adhesion molecule-1 (VCAM1), intercellular adhesion molecule-1 (ICAM1), and E-selectin (E-sel) was increased, ROS levels increased, and superoxide dismutase (SOD) level decreased. Moreover, the protein levels of p-AMPK, Nrf2, and HO-1 were decreased. Compared to the ox-LDL group, SKN treatment improves cell viability, alleviates cell apoptosis and oxidative stress injury, and upregulates the protein levels of p-AMPK, Nrf2, and HO-1. Compound C, si-Nrf2, and si-HO-1 administration inhibits the AMPK/Nrf2/HO-1 signaling pathway, increases ROS generation, and inhibits the antagonistic effect of SKN on ox-LDL-induced HUVECs damage. In summary, SKN suppressed ox-LDL-induced ROS production and improved cell viability and cell apoptosis via the AMPK/Nrf2/HO-1 pathway.