The zinc-fingers and homeoboxes 2 (ZHX2) protein was shown previously to be involved in postnatal repression of α-fetoprotein (AFP) in mice. More recently, loss of ZHX2 expression was often found in human hepatcellular carcinoma (HCC), where AFP is frequently reactivated. Using HepG2 and HepG2.2.15, which express high AFP levels, we show that ZHX2 overexpression significantly decreases of AFP secretion in a dose dependent manner. Furthermore, using LO2 and SMMC7721 cells, which express low AFP levels, we use siRNA inhibition to show that AFP is de-repressed when ZHX2 levels are reduced. This represents the first direct evidence that ZHX2 represses AFP. Co-transfections of ZHX2 and AFP-luciferase reporter genes demonstrate ZHX2 repression is governed by the AFP promoter and requires intact HNF1 binding sites. These data support the idea that ZHX2 contributes to AFP repression in the liver after birth and may also be involved in AFP reactivation in liver cancer.