We previously reported that all-trans-retinoic acid (ATRA) induced apoptosis in N-acetylglucosaminyltransferase V (GnT-V) repressed human hepatocarcinoma 7721 (GnT-V-AS/7721) cells via endoplasmic reticulum (ER) stress. In addition to confirming these findings, we further found that ATRA repressed the expression of betaine-homocysteine methyltransferase (BHMT) and cystathionine-b-synthase (CBS), which are key enzymes that are involved in homocysteine metabolism, increased the level of intracellular homocysteine, and decreased the glutathione (GSH) level in GnT-V-AS/7721 cells. To investigate the effect of ATRA on homocysteine metabolism, cells were challenged with exogenous homocysteine. In GnT-V-AS/7721 cells with ATRA treatment, a significant elevation of intracellular homocysteine levels suggests that ATRA perturbs homocysteine metabolism in GnT-V-AS/7721 cells and, therefore, sensitizes the cells to homocysteine-induced ER stress. An obvious increase in the levels of GRP78/Bip protein and spliced XBP1 mRNA were observed. Furthermore, we observed that ATRA blunted the homocysteine-induced increase of GSH only in GnT-V-AS/7721 cells. These results demonstrate that ATRA intensifies ER stress and induces apoptosis in GnT-V-AS/7721 cells by disturbing homocysteine metabolism through the down-regulation of CBS and BHMT, depleting the cellular GSH and, in turn, altering the cellular redox status. In addition, we showed that ATRA did not trigger ER stress, induce apoptosis, or affect homocysteine metabolism in L02 cells, which is a cell type that is derived from normal liver tissue. These results provide support for the hypothesis that ATRA is an anticancer agent.