Activating transcription factor 4 (ATF4/CREB2) is a member of the cyclic-AMP response element-binding (CREB) family. These proteins have been shown to regulate cell proliferation and differentiation in a broad number of tissues during embryo development. Here we report that male ATF4(-/-) mice are subfertile, despite the fact that they produce sufficient sperm and are able to fertilize wild-type eggs in vitro. An analysis of the ejaculatory ducts revealed abnormal constrictions in the lumen of the vas deferens. The lamina propria layer of the vas deferens was significantly thicker in the ATF4(-/-) mice and the cells that make up this layer were rounder and more abundant than in the ATF4(+/+) littermates. The change in the morphology of the lamina propria was associated with sexual maturation. A histologic analysis of the lamina propria revealed a reduction in the production of elastic fibers and interstitial cells of Cajal, as judged by the expression of neuron-specific enolase. These observations predict that ATF4 is required for the normal differentiation of the lamina propria layer of the vas deferens at sexual maturation. The morphology of the ATF4(-/-) lamina propria and the constriction of the lumen are consistent with an obstruction in the vas deferens contributing to the subfertility of the ATF4(-/-) males.