Background: Organic cation transporters (OCT) are responsible for the uptake of a broad spectrum of endogenous and exogenous substrates. Downregulation of OCT is frequently observed in human hepatocellular carcinoma (HCC) and is associated with a poor outcome. The aim of our current study was to elucidate the impact of OCT3 on hepatocarcinogenesis. Methods: Transcriptional and functional loss of OCT was investigated in primary murine hepatocytes, derived from Oct3-knockout (Oct3-/-; FVB.Slc22a3tm1Dpb) and wildtype (WT) mice. Liver tumors were induced in Oct3-/- and WT mice with Diethylnitrosamine and Phenobarbital over 10 months and characterized macroscopically and microscopically. Key survival pathways were investigated by Western Blot analysis. Results: Loss of Oct3-/- in primary hepatocytes resulted in significantly reduced OCT activity determined by [3H]MPP+ uptake in vivo. Furthermore, tumor size and quantity were markedly enhanced in Oct3-/- mice (p < 0.0001). Oct3-/- tumors showed significant higher proliferation (p < 0.0001). Ki-67 and Cyclin D expression were significantly increased in primary Oct3-/- hepatocytes after treatment with the OCT inhibitors quinine or verapamil (p < 0.05). Functional inhibition of OCT by quinine resulted in an activation of c-Jun N-terminal kinase (Jnk), especially in Oct3-/- hepatocytes. Conclusion: Loss of Oct3 leads to enhanced proliferation and hepatocarcinogenesis in vivo.