Previous studies have revealed that high levels of serum homocysteine (Hcy) are closely associated with the development of juvenile and age-related cataracts. An increased concentration of Hcy is likely to induce gene specific demethylation in DNA promoter regions. The aim of the present study was to prevent this demethylation by administering acetyl-l-carnitine (ALCAR) to human lens epithelial cells (HLECs). Different concentrations of Hcy were used to treat HLECs for 3, 6, 12 and 24 h and the findings were used to determine the optimum dose to induce endoplasmic reticulum (ER) stress. Similarly, the concentration of ALCAR was standardized. The production of reactive oxygen species (ROS) and the percentage of cells undergoing cell death were measured. The levels of antioxidants, ER stress-associated proteins, mRNA levels of nuclear factor erythroid-2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1) and promoter DNA methylation of the Keap1 gene were also assessed. Hcy was observed to induce ER stress, produce ROS and lead to cell death. However, administration of ALCAR prevented these effects to a significant degree. Additionally, western blot analysis revealed that ALCAR increased the levels of antioxidant proteins, including catalase, superoxide dismutase, glutathione peroxidase, Nrf2, Keap1 and glutathione. Similarly, the reverse transcription-quantitative polymerase chain reaction experiments on Nrf2 and Keap1, as well as the bisulfite genomic DNA sequencing analysis revealed a preventive effect of ALCAR against Hcy-induced ER stress. The ER stress-induced activation of the unfolded protein response is responsible for demethylation of Keap1 promoter DNA to activate the expression of the Keap1 protein, which then increases the targeting of Nrf2 for proteosomal degradation. This decrease in Nrf2 activity represses the transcription of numerous antioxidant enzyme genes and alters the redox-balance towards lens oxidation. However, treatment with ALCAR led to significant protection from these effects. The present results suggested that ALCAR either prevents or ameliorates the actions of the antioxidant system in HLECs at the level of the protein and the gene. Further advanced studies are required for the development of ALCAR as an anti-cataract agent.