Expression of Snail1 in epithelial cells triggers an epithelial–mesenchymal transition (EMT). Here, we demonstrate that the synthesis of Zeb2, a transcriptional repressor of E-cadherin, is up-regulated after Snail1-induced EMT. Snail1 does not affect the synthesis of Zeb2 mRNA, but prevents the processing of a large intron located in its 5′-untranslated region (UTR). This intron contains an internal ribosome entry site (IRES) necessary for the expression of Zeb2. Maintenance of 5′-UTR Zeb2 intron is dependent on the expression of a natural antisense transcript (NAT) that overlaps the 5′ splice site in the intron. Ectopic overexpression of this NAT in epithelial cells prevents splicing of the Zeb2 5′-UTR, increases the levels of Zeb2 protein, and consequently down-regulates E-cadherin mRNA and protein. The relevance of these results is demonstrated by the strong association between NAT presence and conservation of the 5′-UTR intron in cells that have undergone EMT or in human tumors with low E-cadherin expression. Therefore, the results presented in this article reveal the existence of a NAT capable of activating Zeb2 expression, explain the mechanism involved in this activation, and demonstrate that this NAT regulates E-cadherin expression.