Hesperidin is a plant-derived bioflavonoid with promising antitumor efficacy, though the underlying mechanisms of action remain poorly elucidated. Thus, we evaluated the in vivo chemopreventive effect of hesperidin against diethylnitrosamine (DEN)-induced hepatocarcinogenesis. We demonstrated the modulatory effect of hesperidin on Nrf2/ARE/HO-1, PPARγ and TGF-β1/Smad3 signaling. Hepatocarcinogenesis was initiated with DEN and promoted with carbon tetrachloride (CCl). DEN/CCl-induced rats were treated with 50 and 100 mg/kg hesperidin throughout the experiment. The results revealed that hesperidin significantly reduced circulating liver function marker enzymes, bilirubin, tumor markers and tumor necrosis factor alpha. Hesperidin prevented liver morphological damage, proliferating cell nuclear antigen (PCNA) expression and oxidative stress as evidenced by the reduced lipid peroxidation and enhanced antioxidant defenses. Liver NF-κB and TGF-β1 expression, and Smad3 phosphorylation were significantly up-regulated in DEN/CCl-induced rats. Hesperidin dramatically abolished NF-κB and TGF-β1/Smad3 signaling as well as collagen deposition in the liver of DEN/CCl-induced rats. In addition, hesperidin markedly up-regulated the expression of Nrf2, HO-1 and PPARγ in the liver of DEN/CCl-induced rats. In conclusion, hesperidin can inhibit hepatocarcinogenesis by suppressing oxidative stress, inflammation, cell proliferation, TGF-β1/Smad3 signaling and collagen deposition. These effects are suggested to be mediated by activating Nrf2/ARE/HO-1 and PPARγ pathways.