Stromal interaction molecule 1 (STIM1) is a transmembrane endoplasmic reticulum protein, and it serves as a Ca2+ sensor and activator of store-operated Ca2+ entry (SOCE). We have previously identified STIM1 in the proteome profile of mice neonatal testes, revealing STIM1 to be associated with neonatal testicular development. Here, to further explore the location and function of STIM1 in mice testes, we studied the effect of Stim1 gene knockdown on neonatal testicular development by testicular culture. Our results revealed that STIM1 was primarily located in Sertoli cells. Knockdown of Stim1 gene using morpholino in neonatal testis caused the mislocation of Sertoli cells and loss of germ cells, which were associated with the aberrant reactive oxygen species (ROS) activation, while inhibition of ROS could partly rescue the phenotypes caused by Stim1 gene knockdown. In conclusion, our study suggests that STIM1 can maintain neonatal testicular development by inhibiting ROS activation.