mRNA 3' processing is dynamically regulated spatially and temporally. However, the underlying mechanisms remain poorly understood. CstF64τ is a paralog of the general mRNA 3' processing factor, CstF64, and has been implicated in mediating testis-specific mRNA alternative polyadenylation (APA). However, the functions of CstF64τ in mRNA 3' processing have not been systematically investigated. We carried out a comprehensive characterization of CstF64τ and compared its properties to those of CstF64. In contrast to previous reports, we found that both CstF64 and CstF64τ are widely expressed in mammalian tissues, and their protein levels display tissue-specific variations. We further demonstrated that CstF64 and CstF64τ have highly similar RNA-binding specificities both in vitro and in vivo. CstF64 and CstF64τ modulate one another's expression and play overlapping as well as distinct roles in regulating global APA profiles. Interestingly, protein interactome analyses revealed key differences between CstF64 and CstF64τ, including their interactions with another mRNA 3' processing factor, symplekin. Together, our study of CstF64 and CstF64τ revealed both functional overlap and specificity of these two important mRNA 3' processing factors and provided new insights into the regulatory mechanisms of mRNA 3' processing.