Morphological and functional changes in cells during the epithelial-mesenchymal transition (EMT) process are known to be regulated by alternative splicing. However, only a few splicing factors involved in EMT have been reported and their underlying mechanisms remain largely unknown. Here, we showed that an isoform of tight junction protein 1 (TJP1) lacking exon 20 (TJP1-α-) is predominantly expressed in tumor tissues and in A549 cells during transforming growth factor-β (TGF-β)-induced EMT. RBM47 promoted the inclusion of exon 20 of TJP1, the alternative exon encoding the α-domain, by which RBM47 recognizes to (U)GCAUG in the downstream intronic region of exon 20. We also found that the first RNA recognition motif (RRM) domain of RBM47 is critical in the regulation of alternative splicing and its recognition to pre-mRNA of TJP1. Furthermore, we demonstrated that the TJP1-α- isoform enhances the assembly of actin stress fibers, thereby promoting cellular migration in a wound healing assay. Our results suggest the regulatory mechanism for the alternative splicing of TJP1 pre-mRNA by RBM47 during EMT, providing a basis for studies related to the modulation of EMT via alternative splicing.