Objective This study aims to explore the mechanism through which resveratrol （Res） mitigates homocysteine （Hcy）-induced oxidative damage in mouse pancreatic β-cells by regulating the expression and methylation level of the Toll-like receptor 4 （TLR4） gene. Methods The effects of different concentrations of homocysteine and resveratrol on the viability of mouse pancreatic β-cells were evaluated using the CCK-8 assay. In each group， the levels of malondialdehyde （MDA）， the activity of glutathione peroxidase （GSH-Px）， and the levels of insulin were measured using an MDA kit， a GSH-Px activity kit， and enzyme-linked immunosorbent assay （ELISA）， respectively. The oxidative stress status was assessed through fluorescence staining. The protein and mRNA expression levels of TLR4 in the control group， the Hcy group， and the homocysteine + resveratrol （Hcy + Res） co-treatment group were detected via Western blot and qRT-PCR. The DNA methylation level of TLR4 was determined using nested methylation-specific PCR， and the protein expression of DNMT1 （DNA methyltransferase 1） was measured by Western blot. Small interfering RNA fragments targeting DNMT1 were constructed and transfected into pancreatic β-cells. Subsequently， the TLR4 protein expression was detected by Western blot. The MDA levels， GSH-Px activity， and insulin levels in the DNMT1 knockdown groups were measured using the aforementioned kits. Likewise， small interfering RNA fragments targeting TLR4 were constructed and transfected into pancreatic β-cells， and then the MDA levels， GSH-Px activity， and insulin levels were measured. Results Cell viability decreased after treatment with 100 μmol/L Hcy. In contrast， intervention with 10 μmol/L Res effectively mitigated the Hcy-induced damage to pancreatic β-cells. Results from the MDA assay and fluorescence staining indicated that， when compared with the Control group， the Hcy group had elevated MDA and reactive oxygen species （ROS） levels （P 0.05）， while these levels declined in the Hcy+Res group （P 0.05）. Findings from the GSH-Px activity assay and ELISA demonstrated that， relative to the Control group， the Hcy group had reduced GSH-Px activity and insulin levels （P 0.05）， whereas the Hcy + Res group showed increased levels （P 0.05）. Western blot and qRT-PCR results showed that the expression levels of TLR4 protein and mRNA were significantly higher in the Hcy group than in the Control group （P 0.05）， while they were significantly lower in the Hcy + Res group （P 0.05）. Conversely， results from nested methylation-specific PCR showed that the TLR4 DNA methylation levels displayed an opposite trend. The expression of DNMT1 protein was lower in the Hcy group （P 0.05） and higher in the Hcy + Res group （P 0.05） compared with the Control group. The expression of TLR4 protein was significantly higher in the Hcy + si-DNMT1 group than in the Hcy + si-NC group （P 0.05）， and it was further increased in the Hcy + Res + si-DNMT1 group compared with the Hcy + Res group （P 0.05）. Compared with the Hcy + si-NC group， the MDA levels were significantly elevated in the Hcy + si-DNMT1 group （P 0.05）. Moreover， when compared with the Hcy + Res group， the MDA levels were further increased in the Hcy + Res + si-DNMT1 group （P 0.05）. The changes in GSH-Px activity and insulin levels were opposite to those of MDA. Further knockdown of TLR4 led to a decrease in MDA levels （P 0.05）， and there was an even greater reduction after Res intervention （P 0.05）. Conversely， GSH-Px activity and insulin levels increased （P 0.05）， and there was a further elevation after Res intervention （P 0.05）. Conclusion Resveratrol alleviates the oxidative damage in pancreatic β-cells induced by homocysteine， a process that is associated with alterations in TLR4 expression and its methylation level.