cAMP-dependent PKA (protein kinase A) plays important regulatory roles during mouse spermatogenesis. PKA-mediated signaling has been shown to regulate gene expression, chromatin condensation, capacitation, and motility during sperm development and function, although how PKA is regulated in spatiotemporal manners during spermatogenesis is not fully understood. In the present study, we found that PKA subunit isoforms are expressed and localized differently in meiotic and post-meiotic mouse spermatogenic cells. Regulatory subunit I alpha (RIα) is expressed in spermatocytes and round spermatids, where it is localized diffusely throughout the cytoplasm of cells. During late spermiogenesis, RIα abundance gradually decreases. On the other hand, RIIα is expressed constantly throughout meiotic and post-meiotic stages, and is associated with cytoskeletal structures. Among several AKAPs (A kinase anchoring proteins) expressed in testis, sperm-specific AKAP3 can be found in the cytoplasm of elongating spermatids and interacts with RIα, as demonstrated by both in vivo and in vitro experiments. In mature sperm, AKAP3 is exclusively found in the principal piece of the flagellum, coincident with only RIIα. Mutagenesis experiments further showed that the preferential interactions of AKAP3 with PKA regulatory subunits are mediated by two highly conserved amphipathic peptides located in the N-terminal region of AKAP3. Thus, AKAP3 is a dual-specificity molecule that modulates PKA isotypes in a spatiotemporal manner during mouse spermatogenesis. Mol. Reprod. Dev.