Hyperhomocysteinemia (HHcy) has been shown to induce endothelial dysfunction, an early event in the progression of atherosclerosis. However, the underlying mechanism of endothelial cell injury in HHcy has not been clearly elucidated. In this study, we examined the effect of homocysteine on tribbles‐related protein 3 (TRB3)‐mediated cell‐cycle arrest in human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with homocysteine (0–250 µmol/L) resulted in inhibition of cell proliferation assessed by [³H]‐thymidine incorporation into DNA. Homocysteine induced cell‐cycle arrest in the G1 phase by up‐regulating the protein levels of p27^kip1. Under these conditions, homocysteine did not induce endoplasmic reticulum stress. However, homocysteine up‐regulated the expression of TRB3, thus leading to the dephosphorylation of Akt (Thr308). Knock‐down of endogenous TRB3 using siRNA significantly suppressed the inhibitory effect of homocysteine on the proliferation of HUVECs. Homocysteine‐induced TRB3 expression was mediated by the cAMP/cAMP response element‐binding protein (CREB) pathway. These results demonstrate that TRB3 is a critical molecule in the homocysteine‐mediated cell‐cycle arrest in endothelial cells. J. Cell. Physiol. 226: 2782–2789, 2011. © 2011 Wiley‐Liss, Inc.