SPAG4 is involved in spermatogenesis and is required for sperm head formation, although it is not needed to establish or maintain the general polarity of the sperm head. It is required for anchoring and organisation of the manchette, a microtubule structure present during sperm development. SPAG4 is also required for targeting SUN3, and probably SYNE1, through a probable SUN1:SYNE3 LINC complex to the nuclear envelope, and it contributes to accurate posterior localisation of that complex in the sperm head. It may anchor SUN3 at the nuclear envelope and is involved in maintaining nuclear envelope integrity.

The SPAG4 protein is also linked to the sperm tail. It may assist the organisation and assembly of outer dense fibres (ODFs), a specific structure of the sperm tail. The sperm flagellum has two cytoskeletal structures associated with the axoneme: the outer dense fibres, which surround the axoneme in the midpiece and principal piece, and the fibrous sheath, which surrounds the outer dense fibres in the principal piece. Defects in these structures are associated with abnormal tail morphology, reduced sperm motility and infertility. In the rat, the protein associates with an outer dense fibre protein through a leucine zipper motif and localises to the microtubules of the manchette and axoneme during sperm tail development.

UniProt records SPAG4 in the nuclear inner membrane and nuclear envelope, in the cytoskeleton including the flagellum axoneme, and as a membrane protein. The SPAG4 gene belongs to the HGNC SUN family, hence the alias SUN4; CT127 is another alias. Alternative splicing produces multiple transcript variants that encode different isoforms. Mouse and rat both have a Spag4 ortholog.