The Glypican 3 (Gpc3) gene is expressed abundantly in the fetal liver, is inactive in the normal adult liver, and is frequently reactivated in hepatocellular carcinoma (HCC). This reactivation in HCC has led to considerable interest in Gpc3 as a diagnostic tumor marker and its possible role in tumorigenesis. Despite this interest, the basis for Gpc3 regulation is poorly understood. On the basis of the similarities between Gpc3 and alpha-fetoprotein expression in the liver, we reasoned that common factors might regulate these 2 genes. Here we identify zinc fingers and homeoboxes 2 (Zhx2) as a regulator of Gpc3. Mouse strain–specific differences in adult liver Gpc3 messenger RNA levels and transgenic mouse studies indicate that Zhx2 represses Gpc3 expression in the adult liver. We also demonstrate that Gpc3 is activated in the regenerating liver following a carbon tetrachloride treatment and that the level of Gpc3 induction is controlled by alpha-fetoprotein regulator 2 (Afr2). Conclusion: We show that Zhx2 acts as a repressor of Gpc3 in the adult liver, and this raises the interesting possibility that Zhx2 might also be involved in Gpc3 reactivation in HCC. We also show that Gpc3 is activated in the regenerating liver in an Afr2-dependent manner. Zhx2 and Afr2 represent the first known regulators of Gpc3. (HEPATOLOGY 2007.)