Background: Forkhead box C1 (FOXC1) is an important cancer-associated gene in tumor. PPAR-gamma and C/EBP alpha are both transcriptional regulators involved in tumor development. Objective: We aimed to clarify the function of PPAR-gamma, C/EBP alpha in hepatocellular carcinoma (HCC) and the relationship of PPAR-gamma, C/EBP alpha and FOXC1 in HCC. Methods: Western blotting, immunofluorescent staining, and immunohistochemistry were used to evaluate protein expression. qRT-PCR was used to assess mRNA expression. Co-IP was performed to detect the protein interaction. And ChIP and fluorescent reporter detection were used to determine the binding between protein and FOXC1 promoter. Results: C/EBP alpha could bind to FOXC1 promoter and PPAR-gamma could strengthen C/EBP alpha's function. Expressions of C/EBP alpha and PPAR-gamma were both negatively related to FOXC1 in human HCC tissue. Confocal displayed that C/EBP alpha was co-located with FOXC1 in HepG2 cells. C/EBP alpha could bind to FOXC1 promoter by ChIP. Luciferase activity detection exhibited that C/EBP alpha could inhibit FOXC1 promoter activity, especially FOXC1 promoter from -600 to -300 was the critical binding site. Only PPAR-gamma could not influence luciferase activity but strengthen inhibited effect of C/EBP alpha. Further, the Co-IP displayed that PPAR-gamma could bind to C/EBP alpha. When C/EBP alpha and PPAR-gamma were both high expressed, cell proliferation, migration, invasion, and colony information were inhibited enormously. C/EBP alpha plasmid combined with or without PPAR-gamma agonist MDG548 treatment exhibited a strong tumor inhibition and FOXC1 suppression in mice. Conclusion: Our data establish C/EBP alpha targeting FOXC1 as a potential determinant in the HCC, which supplies a new pathway to treat HCC. However, PPAR-gamma has no effect on FOXC1 expression.