The PAS domain serine/threonine kinase PASKIN, or PAS kinase, links energy flux and protein synthesis in yeast, regulates glycogen synthesis in mammals, and has been implicated in glucose-stimulated insulin production in b-cells of the pancreas. We previously reported that PASKIN is highly expressed in the testis and that PASKIN-deficient mice are normally viable and fertile. By yeast two-hybrid screening, we identified the multifunctional eukaryotic translation elongation factor eEF1A1 as a novel interaction partner of PASKIN. This interaction was mapped to the PAS A and kinase domains of PASKIN. Kinase assays revealed PASKIN auto-phosphorylation and eEF1A1 target phosphorylation. General protein synthesis was not affected by PASKIN over-expression. With newly generated monoclonal antibodies, PASKIN was localized in the spermatogonia of human testis. PASKIN and eEF1A1 showed a remarkably similar localization in the midpiece of the sperm tail, suggesting a translation-independent function of PASKIN-dependent eEF1A1 phosphorylation in sperm cells such as F-actin organization. By peptide and protein array screening, additional phosphorylation targets of PASKIN were identified. Among others, we could confirm the phosphorylation of mammalian glycogen synthase. The identification of these novel PASKIN phosphorylation targets will help to elucidate the physiologic function of PASKIN.