Puf proteins are regulators of diverse eukaryotic processes including stem cell maintenance, organelle biogenesis, oogenesis, neuron function, and memory formation. At the molecular level, Puf proteins promote translational repression and/or degradation of target mRNAs by first interacting with conserved cis-elements in the 3' untranslated region (UTR). Once bound to an mRNA, Puf proteins elicit RNA repression by complex interactions with protein cofactors and regulatory machinery involved in translation and degradation. Recent work has dramatically increased our understanding of the targets of Puf protein regulation, as well as the mechanisms by which Puf proteins recognize and regulate those mRNA targets. Crystal structure analysis of several Puf-RNA complexes has demonstrated that while Puf proteins are extremely conserved in their RNA-binding domains, Pufs attain target specificity by utilizing different structural conformations to recognize 8-10 nt sequences. Puf proteins have also evolved modes of protein interactions that are organism and transcript-specific, yet two common mechanisms of repression have emerged: inhibition of cap-binding events to block translation initiation, and recruitment of the CCR4-POP2-NOT deadenylase complex for poly(A) tail removal. Finally, multiple schemes to regulate Puf protein activity have been identified, including post-translational mechanisms that allow rapid changes in the repression of mRNA targets.