A multicellular organism’s development is coupled with cellular self‐organization, which is regulated by cell–cell interactions and cell–extracellular matrix (ECM) crosstalk. Testicular cells from different species such as mouse, rat, and porcine can self‐organize into seminiferous tubules both in vitro and in vivo, but the understanding of the functional role of the ECM during this process is limited. Here, it is shown that mouse testicular cells encapsulated with the biomaterial Matrigel can self‐organize into seminiferous tubules with blood‐testis barrier (BTB) formation and Leydig cell differentiation. By varying the encapsulation method, a combination of sodium alginate and collagen is used to promote reorganization of seminiferous tubules, which resemble those in vivo. In addition, the self‐organization ability of testicular cells declines with advanced cell age, and those germ cells play a pivotal role in this process. These findings will be helpful to understand the self‐organization process of testicular cells and provide insights for the reconstruction of testes.