Replication stress is caused by the delay or compromise of DNA synthesis in S-phase. The ability for cells to properly respond to this stress determines viability or lethality and is implicated in many human diseases such as cancer. Post-transcriptional regulation plays an important role in the replication stress response to properly modulate RNA levels. Puf4 is a pumilio family RNA-binding protein that facilitates mRNA degradation through binding the pumilio response element in 3’ untranslated regions. Cytoplasmic RNA granules such as stress granules and p-bodies also show critical roles in the stress response through mRNA triage, storage, and decay. Puf proteins, stress granules, and p-bodies are all highly conserved throughout eukaryotic organisms from single-cell yeasts to humans. However, biological function and mRNA targets of Puf4 and these RNA granules during replication stress remains relatively uncharacterized. In this study, we investigated the role of Puf4, stress granules, and p-bodies in response to hydroxyurea-induced replication stress in the fission yeast Schizosaccharomycess pombe. We determined that p-bodies, but not stress granules, form in response to replication stress and that Puf4 plays a role in regulating p-body formation. Moreover, we suggest Puf4 may play a role in regulating the DNA replication checkpoint as misregulation of puf4+ leads to an increase in cut cells, an abnormal septation phenotype where the septum overlays the nucleus, as seen in several checkpoint mutants. Transcriptome profiling of puf4+ mutants identified four novel putative mRNA targets that are degraded by Puf4 during replication stress. Three of the Puf4 mRNA targets, hhf1+, hht1+, and hta2+, encode for histone proteins while dut1+ encodes a deoxyuridine triphosphate nucleotidohydrolase. The deletion of hhf1+ and hht1+ both suppressed the HU sensitivity of puf4Δ, further supporting the relevance of these genes as Puf4 target mRNAs during replication stress. Lastly, we provided evidence that the 14-3-3 protein Rad24 is an activator of Puf4 by potentially causing cytoplasmic retention of Puf4. Overall, this study provides a foundation for Puf4 function in replication stress through identification of novel target mRNAs and characterizing regulatory interactions with Rad24.