Little is known if and how maternal diet affects the liver phospholipid profiles that contribute to non-alcoholic fatty liver disease (NAFLD) development in offspring. We examined NAFLD phenotypes in male offspring mice of either maternal normal-fat diet (NF group), maternal high-fat diet (HF group), ma-ternal methionine supplement (H1S group), or complete one-carbon supplement (H2S group) added to the maternal HF diet during gestation and lactation. HF offspring displayed worsened NAFLD phenotypes induced by post-weaning HF diet, however, maternal one-carbon supplement prevented such outcome. HF offspring also showed a distinct phospholipid profile from the offspring exposed to H1S or H2S diet. Whole genome bisulfite sequencing (WGBS) analysis further identified five pathways involved in phospholipid metabolism altered by different maternal diet interventions. Furthermore, differential methylated regions (DMRs) on Prkca, Dgkh, Plcb1 and Dgki were identified comparing between HF and NF offspring; most of these DMRs were recovered in H2S off-spring. These methylation pattern changes were associated with gene expression changes: HF diet significantly reduced while H1S and H2S diet recovered their levels. Maternal HF diet disrupted offspring phospholipid profiles contributing to worsened hepatic steatosis. The maternal one-carbon supplement prevented such effects, probably through DNA methylation modification.(c) 2022 Elsevier Inc. All rights reserved.