In this study， we characterized the Puf family gene member Puf3 in the malaria parasites and Secondary structure prediction suggested that the RNA-binding domains of the Puf3 proteins consisted of 11 pumilio repeats that were similar to those in the human Puf-A (also known as PUM3) and Puf6 proteins， which are involved in ribosome biogenesis. Neither (Pf) nor (Py) could be genetically disrupted， suggesting they may be essential for the intraerythrocytic developmental cycle. Cellular fractionation of PfPuf3 in the asexual stages revealed preferential partitioning to the nuclear fraction， consistent with nuclear localization of PfPuf3::GFP and PyPuf3::GFP as detected by immunofluorescence. Furthermore， PfPuf3 colocalized with the nucleolar marker PfNop1， demonstrating that PfPuf3 is a nucleolar protein in the asexual stages. We found， however， that PyPuf3 changed its localization from being nucleolar to being present in cytosolic puncta in the mosquito and liver stages， which may reflect alternative functions in these stages. Affinity purification of molecules that associated with a PTP-tagged variant of PfPuf3 revealed 31 proteins associated with the 60S ribosome， and an enrichment of 28S rRNA and internal transcribed spacer 2 sequences. Taken together， these results suggest an essential function for PfPuf3 in ribosomal biogenesis.