SUN domain-containing proteins belong to a novel protein family. To date, several members--SUN1, SUN2, SUN3, and SPAG4--have been identified as nuclear envelope (NE) proteins. In this study, we sought to characterize and define the potential function of SPAG4L, a newly identified SUN protein. Using bioinformatic analysis, we found that SPAG4L contained a conserved SUN domain in the C-terminal. Subcellular localization analysis indicated that the expression of green fluorescent protein-labeled full-length SPAG4L was localized to the NE and the endoplasmic reticulum (ER). Deletion analysis revealed that the transmembrane region and the coiled-coil domain, but not the SUN domain, were required for localization of SPAG4L to the NE and ER. Subsequently, we confirmed that the human testes expressed endogenous SPAG4L as a 43-kDa protein. Further studies revealed that mouse Spag4L colocalized with the NE marker Lamin B1 and the ER marker PDI in isolated mouse spermatocytes. In addition, the expression of Spag4L was observed in meiosis I and II stages, suggesting that Spag4L may be involved in NE reconstitution and nuclear migration occurring during the process of spermatocyte division. Together, the findings indicate that SPAG4L, a new NE protein, may play an important role in the meiotic stage of spermatogenesis.