Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis. In a manner comparable to hematopoietic stem cell transplantation, SSCs colonize the niche of recipient testes and reinitiate spermatogenesis following microinjection into the seminiferous tubules. However, little is known about the homing mechanism of SSCs. Here we examined the role of adhesion molecules in SSC homing. SSCs isolated from mice carrying loxP-tagged beta1-integrin alleles were ablated for beta1-integrin expression by in vitro adenoviral cre transduction. The beta1-integrin mutant SSCs showed significantly reduced ability to recolonize recipient testes in vivo and to attach to laminin molecules in vitro. In contrast, genetic ablation of E-cadherin did not impair homing, and E-cadherin mutant SSCs completed normal spermatogenesis. In addition, the deletion of beta1-integrin on Sertoli cells reduced SSC homing. These results identify beta1-integrin as an essential adhesion receptor for SSC homing and its association with laminin is critical in multiple steps of SSC homing.