Homeostasis and maturation of the mammalian intestinal epithelium are preserved through strict regulation of cell proliferation, apoptosis and differentiation, but the exact mechanism underlying this process remains largely unknown. c-Jun N-terminal kinase 2 (JNK2) is highly expressed in the intestinal mucosa and its activation plays an important role in proliferation and also mediates apoptosis in cultured intestinal epithelial cells (IECs). Here, we investigated the in vivo function of JNK2 in the regulation of intestinal epithelial homeostasis and maturation by using a targeted gene deletion approach. Targeted deletion of the jnk2 gene increased cell proliferation within the crypts in the small intestine and disrupted mucosal maturation as indicated by decreases in the height of villi and villus/crypt ratio. JNK2 deletion also decreased susceptibility of the intestinal epithelium to apoptosis. JNK2-deficient intestinal epithelium was associated with an increase in the level of the RNA-binding protein HuR and with a decrease in the abundance of CUG-binding protein 1 (CUGBP1). In studies in vitro, JNK2 silencing protected IEC-6 cells against apoptosis and this protection was prevented by inhibiting HuR. Ectopic overexpression of CUGBP1 repressed IEC-6 cell proliferation, whereas CUGBP1 silencing enhanced cell growth. These results indicate that JNK2 is essential for maintenance of normal intestinal epithelial homeostasis and maturation under biological conditions by differentially modulating HuR and CUGBP1.