We previously found that Homeobox containing 1 (HMBOX1) was required for bone mesenchymal stem cell (BMSC) and mouse embryonic stem cell (ESC) differentiation into vascular endothelial cells (VECs). However, the function of HMBOX1 in VECs is still unknown. In this study, we found that HMBOX1 was abundantly expressed in the cytoplasm of human umbilical vascular endothelial cells (HUVECs). Knockdown of HMBOX1 induced apoptosis and inhibited autophagy. Overexpression of HMBOX1 inhibited apoptosis induced by fibroblast growth factor 2 deprivation and promoted autophagy. Metallothionein 2A (MT2A) was identified as an interaction protein with HMBOX1 by yeast two-hybrid assay and confirmed by co-immunoprecipitation. Overexpression of HMBOX1 elevated intracellular free zinc level. Knockdown of MT2A inhibited this phenomenon. Moreover, N,N,N = ,N = -tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a zinc chelator, reversed the anti-apoptosis and pro-autophagy effects of HMBOX1. In conclusion, HMBOX1 regulated intracellular free zinc level by interacting with MT2A to inhibit apoptosis and promote autophagy in VECs.