Although terminal differentiation of intestinal epithelium is essential for the efficient digestion and absorption of nutrients, little is known about the molecular mechanisms underlying this process. Recent studies have shown that Elf3 (E74-like factor 3), a member of the ETS transcription factor family, has an essential role in the terminal differentiation of absorptive enterocytes and mucus-secreting goblet cells. Here, we demonstrated that Crif1 (CR6-interacting factor 1) functions as transcriptional coactivator of Elf3 in intestinal epithelium differentiation. The intestinal epithelium-specific Crif1-deficient mice died soon after birth and displayed severe alterations of tissue architecture in the small intestine, including poor microvillus formation and abnormal differentiation of absorptive enterocytes. Strikingly, these phenotypes are largely similar to that of Elf3-deficient mice, suggesting that Elf3 signaling in the intestinal epithelium depends on the Crif1 expression. We dissected this relationship further and found that Crif1 indeed interacted with Elf3 through its ETS DNA binding domain and enhanced the transcriptional activity of Elf3 by regulating the DNA binding activity. Knockdown of Crif1 by RNA interference conversely attenuated the transcriptional activity of Elf3. Consistently, the expression level of Tgf-betaRII (transforming growth factor beta type II receptor), a critical target gene of Elf3, was dramatically reduced in the Crif1-deficient mice. Our results reveal that Crif1 is a novel and essential transcriptional coactivator of Elf3 for the terminal differentiation of absorptive enterocytes.