Laminins are important for basement membrane structure and function. The laminin α2 chain is a major component of muscle basement membranes, and mutations in the laminin α2 gene lead to congenital muscular dystrophy in humans and mice. Although the laminin α2 chain is prominently expressed in testicular basement membranes, its role in testis has remained unclear. Here, we show that laminin α1, α2, β1, β2, γ1, and γ3 chains are the major laminin chains in basement membranes of seminiferous tubules. In laminin α2 chain-deficient dy3K/dy3K mice, lack of laminin α2 chain led to concurrent reduction of laminin γ3 chain and abnormal testicular basement membranes. Seminiferous tubules of laminin α2 chain-deficient dy3K/dy3K mice displayed a defect in the timing of lumen formation, resulting in production of fewer spermatides. We also demonstrate that overexpression of laminin α1 chain in testis of dy3K/dy3K mice compensated for laminin α2 chain deficiency and significantly reversed the appearance of the histopathological features. We thus provide genetic data that laminin α chains are essential for normal testicular function in vivo.