Betaine, which donates methyl groups through methionine metabolism for DNA and protein methylation, is critical for epigenetic gene regulation especially during fetal development. Here we fed gestational sows (8 per group) with control or betaine supplemented diets (3 g/kg, from the first day of gestation to parturition) to explore the effects of maternal betaine on hepatic cell proliferation in neonatal piglets. Newborn piglets were individually weighed immediately after birth and the piglets from the same litter were kept together in the warm creep area. One male piglets of the mean body weight were selected per litter and killed for tissue sampling before suckling. Neonatal piglets born to betaine-supplemented sows demonstrated a reduction of cell number and DNA content (P < 0.05) in the liver, which was associated with significantly downregulated hepatic expression of cell cycle regulatory genes (P < 0.05), cyclin 2 (CCND2) and presenilin1 (PSEN1). Moreover, STAT3 binding to the promoter of CCDN2 and PSEN1genes was lower (P < 0.05) in betaine-exposed piglets, which was accompanied by strong reduction (P < 0.05) of STAT3 mRNA and protein expression along with its phosphorylation at Tyr⁷⁰⁵ and Ser⁷²⁷ residues (P < 0.05). Also, prenatal betaine exposure significantly attenuated upstream kinases (P < 0.05) of STAT3 signaling pathway (phospho-ERK1/2, phospho-SRC and phospho-JAK2) in the liver of neonates. Furthermore, the repressed STAT3 expression in the liver of betaine-exposed piglets was associated with DNA hypermethylation (P < 0.05) and more enriched repression histone mark H3K27me3 (P < 0.05) on its promoter, together with significantly upregulated expression (P < 0.05) of H3K27me3 and enhancer of zeste homolog 2 (EZH2) protein, as well as miR-124a (P < 0.05) which targets STAT3. Taken together, our results suggest that maternal dietary betaine supplementation during gestation inhibits cell proliferation in the liver of neonatal piglets through epigenetic regulation of hepatic CCDN2 and PSEN1 genes via a STAT3-dependent pathway.