Maternal consumption of a high-fat diet has been linked to increased risks of obesity and impaired glucose metabolism in offspring. However, the precise epigenetic mechanisms governing these intergenerational effects, particularly during the early stages of offspring development, remain poorly understood. In this study, female C57BL/6J mice were randomly assigned to either a high-fat diet or normal chow diet throughout gestation and lactation. Methylated DNA immunoprecipitation (MeDIP) coupled with microarray analysis was employed to identify differentially methylated genes in the livers of offspring at weaning age. We found that maternal high-fat diet feeding predisposes offspring to obesity and impaired glucose metabolism as early as the weaning period. DNA methylation profile analysis unveiled a significant enrichment of differentially methylated genes within the natural killer (NK) cell-mediated cytotoxicity pathway. MeDIP-PCR validated reduced methylation levels of specific genes within this pathway, including tumour necrosis factor α (TNF-α), phosphoinositide 3-kinase (PI3K), and SHC adaptor protein 1 (SHC1). Consistently, the expressions of TNF-α, PI3K, and SHC1 were significantly upregulated, accompanied by elevated serum TNF-α and interleukin-6 (IL-6) levels in offspring from dams fed with high-fat diet. Moreover, we assessed the expressions of genes associated with NK cell activities, uncovering a notable rise in hepatic granzyme B levels and a trend towards increased CD107a expression in offspring from dams fed a high-fat diet. In addition, methylation levels of TNF-α, PI3K, and SHC1 promoters were inversely correlated with glucose response during glucose tolerance testing. In conclusion, our findings underscore the critical role of the NK cell-mediated cytotoxicity signaling pathway in mediating DNA methylation patterns, thereby contributing to the programming effects of maternal high-fat diet consumption on offspring glucose metabolism as early as the weaning period.