The vas deferens is critical for the transport of sperm and can be the site of obstruction resulting from inflammation, infections or following vasectomy. Recently, we demonstrated that the cytokine, activin A, displays a pronounced proximal to distal gradient of expression in the epididymis and vas. Conversely, follistatin, the activin-binding protein, is highly expressed in the vas with low epididymal expression. The roles of these proteins in the vas have not been studied to date. This study investigated the structure and localisation of activin in the vas of wild-type and follistatin 288-deficient transgenic mice, in which the Fst gene has been deleted and replaced by a human FST315 construct (tghFST315 mice). In contrast to the wild-type, the proximal region of the vas of tghFST315 mice displays coiling. This region showed structural defects including a thinner lamina propria and increased apoptosis of epithelial cells. Immunohistochemistry localised both activin A and B to the epithelial cells of the vas, with negligible staining in the lamina propria and stroma. The pattern of activin A staining intensity in the epithelium matched the declining gene expression pattern along the length of the vas. Activin A expression was localised to the cytoplasm and stereocilia of the epithelial cells, and to sperm in the lumen. The muscle of the vas showed weak staining for activin B, but not activin A. Enhanced activin A staining was observed in epithelial cells of the tghFST315 mouse when compared to wild-type. By contrast, activin B staining was more prominent in the wild-type epithelium. In conclusion, these data establish the epithelial cells as the source of activin A and B in the vas. As coiling in the tghFST315 vas was associated with increased activin A and reduced activin B in the epithelium, this indicates a role for follistatin in regulating vas structure/function.