ABSTRACT Actin-related protein T3 (ACTRT3) is localized in the perinuclear theca (PT) of murine spermatids. We generated Actrt3−/− male mice and showed that they are subfertile, with defects of the acrosome first observed during cap phase. Actrt3 deficiency causes reduced protein levels of the trans-Golgi network markers TGN46 and GOPC and mislocalization of the cis-Golgi protein GM130. Reduction of the autophagy markers LC3B, CTSB and mTOR indicates that loss of ACTRT3 leads to impaired Golgi trafficking and autophagic flux, which are required for acrosome biogenesis. In addition, levels of PFN3, a protein involved in acrosome biogenesis, were significantly reduced. Further, co-immunoprecipitation revealed interaction of ACTRT3 with the PT proteins ACTRT1, ACTRT2, ACTL7A, SPEM2 and the sperm surface protein ZPBP. This suggested that ACTRT3 is a part of the complex 3D scaffold of the PT and contributes to ZPBP localization. Mass spectrometry revealed enrichment of cytoskeletal regulators such as CFL1 and CNN1. Expression of Actrt3 caused changes in HEK239T cell shape and F-actin filament distribution, suggesting a role in cytoskeletal shaping. We conclude that lack of ACTRT3 affects acrosome biogenesis, PT structure and actin remodeling.