The physiological regulation of Oestrogen receptor α (ERα) and peroxisome proliferator‐activated receptor alpha (PPARα) in Hepatocellular carcinoma (HCC) remains unknown. The present study we first treat the cells with fenofibrate and further investigated the possible mechanisms of 17β‐estradiol (E₂) and/or ERα on regulating PPARα expression. We also found higher PPARα expression in the tumor area than adjacent areas and subsequently compared PPARα expression in four different hepatic cancer cell lines. Hep3B cells were found to express more PPARα than the other cell lines. Using the PPARα agonist fenofibrate, we found that fenofibrate increased Hep3B cell proliferation efficiency by increasing cell cycle proteins, such as cyclin D1 and PCNA, and inhibiting p27 and caspase 3 expressions. Next, we performed transient transfections and immuno‐precipitation studies using the pTRE2/ERα plasmid to evaluate the interaction between ERα and PPARα. ERα interacted directly with PPARα and negatively regulated its function. Moreover, in Tet‐on ERα over‐expressed Hep3B cells, E₂ treatment inhibited PPARα, its downstream gene acyl‐CoA oxidase (ACO), cyclin D1 and PCNA expression and further increased p27 and caspase 3 expressions. However, over‐expressed ERα plus 17‐β‐estradiol (10⁻⁸ M) reversed the fenofibrate effect and induced apoptosis, which was blocked in ICI/melatonin/fenofibrate‐treated cells. This study illustrates that PPARα expression and function were negatively regulated by ERα expression in Hep3B cells.