Background: Hepatocyte nuclear factor 4 alpha (HNF4 alpha) plays an important role in tumourigenesis. There is growing evidence indicating that HNF4 alpha transcribed by promoter 1 (P1-HNF4 alpha) is expressed at relatively low levels in HCC and its presence predicts a favourable outcome for hepatocellular carcinoma (HCC) patients. However, the role of HNF4 alpha transcribed by promoter 2 (P2-HNF4 alpha) in HCC remains unclear. Methods: A total of 615 HCC specimens were obtained to construct tissue microarrays and perform immunohistochemistry. The relationship between P2-HNF4 alpha and clinical features of HCC patients were analysed. Kaplan-Meier analysis was conducted to assess the prognostic value of P2-HNF4 alpha. Results: The results showed that the expression of P2-HNF4 alpha in HCC was noticeably increased in HCC tissues compared with the nontumourous tissues. In addition, P1-HNF4 alpha expression was negatively correlated with P2-HNF4 alpha expression (p=0.023). High P2-HNF4 alpha expression was significantly associated with poor differentiation of HCC (p = 0.002) and vascular invasion (p =0.017). Kaplan-Meier analysis showed that P2-HNF4 alpha expression was closely correlated with overall survival in the training group (p = 0.01), validation group (p =0.034), and overall group of patients with HCC (p < 0.001). Conclusions: Our data show that the role of HNF4 alpha in cancer development needs to be further refined. P2-HNF4 alpha, different from P1-HNF4 alpha, is markedly upregulated and serves as an oncogene-associated protein in HCC. Our study therefore provides a promising biomarker for prognostic prediction and a potential therapeutic target for HCC.