Activin A signaling stimulates proliferation and inhibits differentiation of testicular Sertoli cells throughout development. At puberty, activin levels decline as Sertoli cells undergo what has been accepted to be a process of ‘terminal’ differentiation in order to sustain spermatogenesis in the adult. Characteristics of differentiated Sertoli cells include cessation of proliferation, formation of tight junctions, and expression of mature protein markers. In this study, we aimed to determine whether activin A could cause differentiated Sertoli cells to revert to a functionally immature phenotype. To increase systemic activin levels in vivo, the right tibialis anterior muscle of seven-week old C57BL/6 mice was transduced with an adeno-associated virus (rAAV6) expressing activin A. After 4 weeks, serum activin A was elevated ~4-fold while testis mass was reduced by 23.5% compared with control animals. Morphologically, activin A-treated testes displayed a hypo-spermatogenic phenotype, consistent with a failure of Sertoli cells to support spermatogenesis. Blood-testis barrier function was ablated in some tubules as assessed using a permeability tracer. Additionally, activin A potently antagonized tight junctions in a model of primary differentiated rat Sertoli cells in vitro, and re-initiated Sertoli cell proliferation as determined by 5-ethynyl-2´-deoxyuridine (EdU) incorporation. Activin-stimulated proliferative Sertoli cells were found to re-express juvenile markers, including cytokeratin-18 (increased 18-fold compared with untreated cells), suppress mature markers, including claudin-11 (0.23-fold), and up-regulate proliferative markers (cyclin D2, 6.6-fold). We conclude that activin A is the first identified factor capable of re-programming differentiated Sertoli cells to an immature, de-differentiated phenotype. This study indicates that activin signalling must be strictly controlled in the adult in order to maintain Sertoli cell function in spermatogenesis.