The mitogen-activated protein kinase (MAPK) p38α controls inflammatory responses and cell proliferation. Using mice carrying conditional Mapk14 (also known as p38α) alleles, we investigated its function in postnatal development and tumorigenesis. When we specifically deleted Mapk14 in the mouse embryo, fetuses developed to term but died shortly after birth, probably owing to lung dysfunction. Fetal hematopoietic cells and embryonic fibroblasts deficient in p38α showed increased proliferation resulting from sustained activation of the c-Jun N-terminal kinase (JNK)–c-Jun pathway. Notably, in chemical-induced liver cancer development, mice with liver-specific deletion of Mapk14 showed enhanced hepatocyte proliferation and tumor development that correlated with upregulation of the JNK–c-Jun pathway. Furthermore, inactivation of JNK or c-Jun suppressed the increased proliferation of Mapk14-deficient hepatocytes and tumor cells. These results demonstrate a new mechanism whereby p38α negatively regulates cell proliferation by antagonizing the JNK–c-Jun pathway in multiple cell types and in liver cancer development.