We recently reported that silencing of the polyadenylation factor EhCFIm25 in Entamoeba histolytica, the protozoan which causes human amoebiasis, affects trophozoite proliferation, death, and virulence, suggesting that EhCFIm25 may have potential as a new biochemical target. Here, we performed a shotgun proteomic analysis to identify modulated proteins that could explain this phenotype. Data are available via ProteomeXchange with identifier PXD027784. Our results revealed changes in the abundance of 75 proteins. Interestingly, STRING analysis, functional GO-term annotations, KEGG analyses and literature review showed that modulated proteins are mainly related to glycolysis and carbon metabolism, cytoskeleton dynamics and parasite virulence, as well as gene expression and protein modifications. Further studies are needed to confirm the hypotheses emerging from this proteomic analysis, to thereby acquire a comprehensive view of the molecular mechanisms involved.