Recent studies of the Puf family of RNA-binding proteins revealed that besides their traditional roles in translational regulation of mRNAs, some Puf proteins are also involved in ribosome biogenesis by binding rRNA. In this study, we report the role of a Puf-like protein (Puf3) in ribosome biogenesis in Plasmodium falciparum and Plasmodium yoelii. Secondary structure prediction suggested that the RNA-binding domain of Puf3 consists of 11 pumilio repeats, similar to human Puf-A/yeast Puf6, which is involved in ribosome biogenesis. Neither pfpuf3 nor pypuf3 could be genetically disrupted, suggesting they may be essential for the intraerythrocytic developmental cycle (IDC). A time-course study of PfPuf3 protein indicated that PfPuf3 was expressed during the entire IDC, with peak expression in early trophozoites. Cellular fractionation of PfPuf3 revealed that it is preferentially partitioned to the nuclear rather than the cytoplasmic fractions, which is consistent with a nuclear localization of PfPuf3::GFP and PyPuf3::GFP as seen by immunofluorescence. Further, we found PfPuf3 co-localized with a well-known nucleolus maker, PfNop1, demonstrating that PfPuf3 is a nucleolar protein. This localization is in contrast to the cytoplasmic localization of PfPuf1 and PfPuf2, but matches the localization of human Puf-A and yeast Puf6. Affinity purification of a C-terminal PTP-tagged variant of PfPuf3 revealed an association with 32 proteins associated with the 60S ribosome, and an enrichment of 28S rRNA and internal transcribed spacer 2. Taken together, these results demonstrate a nucleolar localization of PfPuf3 and suggest an essential function of PfPuf3 in ribosomal biogenesis.