Induction of endothelial cytotoxicity by hyperglycemia in diabetes has been widely accepted. Emodin is a natural anthraquinone in rhubarb used for treatment of diabetes, but its mechanism of action is not fully understood. This study aimed to examine the potential beneficial effects of emodin on endothelial cytotoxicity caused by high glucose milieu. Culture of human umbilical vein endothelial cells (HUVECs) with high concentrations of glucose resulted in damage to the cells, leading to decreased formazan products by 14–27%, reduced DNA contents by 12–19%, and increased hypodiploid apoptosis by 40–109%. These adverse effects of high glucose could be prevented to a large extent by co-culture with 3 μM of emodin which per se did not affect HUVECs viability. In addition, CCL5 expression of HUVECs cultured in high glucose medium was significantly elevated at both mRNA and protein levels, an effect abolished after treatment with emodin. Moreover, the enhanced adhesion of monocytes to HUVECs (2.1–2.2 fold over control) and elevated chemotaxis activities (2.3–2.4 fold over control) in HUVECs cultured in high glucose medium were completely reversed by emodin. Emodin also suppressed activation of p38 MAPK and ERK1/2 due to high glucose. Our data demonstrated that endothelial cytotoxicity occurred clearly when HUVECs were exposed to high glucose milieu and emodin was able to alleviate the impairments. The protective effects of emodin might be related to the inhibition of CCL5 expression and subsequent cell stress/inflammatory events possibly mediated by activation of MAPK signaling pathways.