Glypican 3 (GPC3) belongs to a family of glycosylphosphatidylinositol‐anchored, cell‐surface heparan sulfate proteoglycans. GPC3 is overexpressed in hepatocellular carcinoma. Loss‐of‐function mutations of GPC3 result in Simpson‐Golabi‐Behmel syndrome, an X‐linked disorder characterized by overgrowth of multiple organs, including the liver. Our previous study showed that GPC3 plays a negative regulatory role in hepatocyte proliferation, and this effect may involve CD81, a cell membrane tetraspanin. To further investigate GPC3 in vivo, we engineered transgenic (TG) mice overexpressing GPC3 in the liver under the control of the albumin promoter. GPC3 TG mice with hepatocyte‐targeted, overexpressed GPC3 developed normally in comparison with their nontransgenic littermates but had a suppressed rate of hepatocyte proliferation and liver regeneration after partial hepatectomy. Moreover, gene array analysis revealed a series of changes in the gene expression profiles in TG mice (both in normal mice and during liver regeneration). In unoperated GPC3 TG mice, there was overexpression of runt related transcription factor 3 (7.6‐fold), CCAAT/enhancer binding protein alpha (2.5‐fold), GABA A receptor (2.9‐fold), and wingless‐related MMTV integration site 7B (2.8‐fold). There was down‐regulation of insulin‐like growth factor binding protein 1 (8.4‐fold), Rab2 (5.6‐fold), beta‐catenin (1.7‐fold), transforming growth factor beta type I (3.1‐fold), nodal (1.8‐fold), and yes‐associated protein (1.4‐fold). Changes after hepatectomy included decreased expression in several cell cycle–related genes. Conclusion: Our results indicate that in GPC3 TG mice, hepatocyte overexpression of GPC3 suppresses hepatocyte proliferation and liver regeneration and alters gene expression profiles, and potential cell cycle–related proteins and multiple other pathways are involved and affected. (HEPATOLOGY 2010;52:1060–1067)