Acephalic spermatozoa syndrome (ASS) is a severe teratospermia with decaudated, decapitated and malformed sperm, resulting in male infertility. Nuclear envelop (NE) protein SUN5 localizes to the junction between the sperm head and tail. Mutations in SUN5 gene have been identified most frequently (33-47%) in ASS cases, and the molecular mechanism needs to be explored. In the present study, we generated Sun5 knockout mice, which presented the phenotype of ASS. Nuclear membrane protein LaminB1, cytoskeletal GTPases Septin12 and Septin2 were identified as potential partners for interacting with SUN5 in mouse testis by immunoprecipitation-mass spectrometry (IP-MS). Further studies demonstrated that SUN5 co-operated with LaminB1, and connected proximal centriole by interacting with Septin12. The binding between SUN5 and Septin12 promoted the aggregation of SUN5 and Septin12 in the sperm neck. The disruption of LaminB1/SUN5/Septin12 complex by Sun5 deficiency caused separation of the Septin12- proximal centriole from the nucleus, leading to the breakage of the head-to-tail junction. Collectively, these data provide new insights into the pathogenesis of ASS caused by SUN5 deficiency.