Mutant mice lacking a testis-specific cytoplasmic poly(A) polymerase, PAPOLB/TPAP, exhibit spermiogenesis arrest and male infertility. However, the mechanism by which PAPOLB regulates spermiogenesis remains unclear. In this study, we examined the relationships between PAPOLB and other spermiogenesis regulators present in the chromatoid body (CB). The loss of PAPOLB had no impact either on the abundance of CB components such as PIWIL1, TDRD6, YBX2, and piRNAs, or on retrotransposon expression. In addition, localization of CB proteins and CB architecture were both normal in PAPOLB-null mice. No interactions were observed between PAPOLB and PIWIL1 or YBX2. While PIWIL1 and YBX2 were associated with translationally inactive messenger ribonucleoproteins and translating polyribosomes, PAPOLB was present almost exclusively in the mRNA-free fractions of sucrose gradients. These results suggest that PAPOLB may regulate spermiogenesis through a pathway distinct from that mediated by CB-associated factors.