Proteins may undergo a type of posttranslational modification – polyglutamylation, where a glutamate residue is enzymatically linked to the γ-carboxyl group of a glutamate in the primary sequence of proteins and additional glutamates are then sequentially added via α-carboxyl–linkages to the growing glutamate side chain. Nna1 (a.k.a. CCP1) defines the 6-member cytosolic carboxypeptidase (CCP) family that metabolizes polyglutamate side chain and its loss results in neurodegeneration and male infertility. Whereas most CCPs catalyze hydrolysis of α-carboxyl-linked glutamates, CCP5 uniquely metabolizes the γ-carboxyl linked, branch point glutamate. Using purified recombinant mouse CCP5, we confirmed that it metabolized γ-carboxyl-linked glutamate of synthetic substrates and tubulin. Despite this unique feature and its indispensible functions in lower species, we found that unlike Nna1, CCP5 is not essential for neuronal survival in mouse. CCP5 deficiency does cause male infertility. However, the mechanism by which this occurs is distinct from that of Nna1 loss. Instead, it is phenotypically reminiscent of the infertility of olt mice. Our findings suggest that Nna1 and CCP5 do not work coordinately in the same pathway in either the nervous system or spermatogenesis. This is the first study addressing the function of CCP5 in mammals.