Aim. To investigate the relationship between alpha-fetoprotein and zinc fingers and homeoboxes 2 in hepatocellular carcinoma. Materials and Methods. The expressions of zinc fingers and homeoboxes 2, nuclear factor-YA, and alpha-fetoprotein mRNA in 63 hepatocellular carcinoma were detected by reverse transcriptase-polymerase chain reaction and compared with the clinical parameters of the patients. Selectively, silence of zinc fingers and homeoboxes 2 in HepG2 cells was detected by RNA interference technique. Results. Alpha-fetoprotein mRNA expression was detected in 60.3% of hepatocellular carcinoma cases. Zinc fingers and homeoboxes 2 mRNA expression (36.5%) was significantly negatively correlated with serum alpha-fetoprotein concentration and mRNA expression. A strong positive correlation was found between zinc fingers and homeoboxes 2 and nuclear factor-YA mRNA expression (42.9%), while the latter was negatively correlated with serum alpha-fetoprotein concentration and mRNA expression. Treatment with zinc fingers and homeoboxes 2 small interfering RNA led to 85% and 83% silence of zinc fingers and homeoboxes 2 mRNA and protein expression and 60% and 61% reduction of nuclear factor-YA mRNA and protein levels in the HepG2 cells, respectively. Downregulation of zinc fingers and homeoboxes 2 also induced a 2.4-fold increase in both alpha-fetoprotein mRNA and protein levels. Conclusions. Zinc fingers and homeoboxes 2 can regulate alpha-fetoprotein expression via the interaction with nuclear factor-YA in human hepatocellular carcinoma and may be used as an adjuvant diagnostic marker for alpha-fetoprotein-negative hepatocellular carcinoma.