JNK proteins have been shown to be involved in liver carcinogenesis in mice, but the extent of their involvement in the development of human liver cancers is unknown. Here, we show that activation of JNK1 but not JNK2 was increased in human primary hepatocellular carcinomas (HCCs). Further, JNK1 was required for human HCC cell proliferation in vitro and tumorigenesis after xenotransplantation. Importantly, mice lacking JNK1 displayed decreased tumor cell proliferation in a mouse model of liver carcinogenesis and decreased hepatocyte proliferation in a mouse model of liver regeneration. In both cases, impaired proliferation was caused by increased expression of p21, a cell-cycle inhibitor, and reduced expression of c-Myc, a negative regulator of p21. Genetic inactivation of p21 in JNK1-/- mice restored hepatocyte proliferation in models of both liver carcinogenesis and liver regeneration, and overexpression of c-Myc increased proliferation of JNK1-/- liver cells. Similarly, JNK1 was found to control the proliferation of human HCC cells by affecting p21 and c-Myc expression. Pharmacologic inhibition of JNK reduced the growth of both xenografted human HCC cells and chemically induced mouse liver cancers. These findings provide a mechanistic link between JNK activity and liver cell proliferation via p21 and c-Myc and suggest JNK targeting can be considered as a new therapeutic approach for HCC treatment.