<p>Protein dephosphorylation mediated by phosphatases regulates spermatogenesis. However, which proteins are dephosphorylated and how they regulate spermatogenesis are largely unknown. Here, we show that germline-specific deletion of protein phosphatase 6 regulatory subunit 3 (PPP6R3), which determines substrate specificity of protein phosphatase 6 (PP6), causes abnormal spermatogonial differentiation and male infertility, accompanied by translation inhibition. PPP6R3 directly interacts with EIF3C and EIF4G1 in KIT⁺ spermatogonia. Decreased levels of non-phosphorylated EIF3C and EIF4G1 after PPP6R3 deletion attenuate their enrichment for mRNAs associated with spermatogonial differentiation, and increased phosphorylation levels promote their degradation. Specifically, the phosphorylation status both of EIF3C^S39 and EIF4G1^S1217 are significantly up-regulated in mutant mice. Overexpression of EIF3C^S39A and EIF4G1^S1217A mutants in <i>Ppp6r3</i>-cKO spermatogonial progenitor cells compensates for the deficiency of differentiation potential by upregulating translation rates of differentiation-associated mRNAs. Our findings demonstrate EIF3C and EIF4G1, as specific substrates of PPP6R3/PP6 holoenzyme, are required for translation activation during spermatogonial differentiation.</p>