AbstractSeminiferous tubules (STs) in the mammalian testes are connected to the rete testis (RT) via a Sertoli valve (SV). Spermatozoa produced in the STs are released into the tubular luminal fluid and passively transported through the SV into the RT. However, the physiological functions of the RT and SV remain unclear. Here, we identified the expression ofSox17in RT epithelia. The SV valve was disrupted before puberty in RT-specificSox17conditional knockout (Sox17-cKO) male mice. This induced a backflow of RT fluid into the STs, which caused aberrant detachment of immature spermatids. RT ofSox17-cKO mice had reduced expression levels of various growth factor genes, which presumably support SV formation. When transplanted next to theSox17+RT, Sertoli cells ofSox17-cKO mice reconstructed the SV and supported proper spermiogenesis in the STs. This study highlights the novel and unexpected modulatory roles of the RT in SV valve formation and spermatogenesis in mouse testes, as a downstream action ofSox17.