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We investigated the nutritional effects on carcass traits, gene expression and DNA methylation in a three generation Large White pig feeding experiment. A group of experimental (E) F0 boars were fed a standard diet supplemented with high amounts of methylating micronutrients whereas a control group (C) of F0 boars received a standard diet. These differentially fed F0 boars sired F1 boars which then sired 60 F2 pigs. Carcass traits were compared between 36 F2 descendants of E F0 boars and 24 F2 descendants of C F0 boars. The two F2 offspring groups differed with respect to backfat percentage (P = 0.03) and tended to differ with respect to adipose tissue (P = 0.09), fat thickness at the 10(th) rib (P = 0.08) and at the croup (P = 0.09) as well as percentages of shoulder (P = 0.07). Offspring from the experimental F0 boars had a higher percentage of shoulder and were leaner compared to the control group. Gene expression profiles showed significant twofold differences in mRNA level between 8 C F2 offspring and 8 E F2 offspring for 79, 64 and 53 genes for muscle, liver and kidney RNA, respectively. We found that in liver and muscle respective pathways of lipid metabolism and metabolic pathway were over-represented for the differentially expressed genes between these groups. A DNA methylation analysis in promoters of differentially expressed genes indicated a significant difference in DNA methylation at the IYD gene. If these responses on carcass traits, gene expression and DNA methylation withstand verification and can indeed be attributed to transgenerational epigenetic inheritance, it would open up pioneering application in pork production and would have implications for human health.