Malaria, a disease caused by infection with parasites of the genus Plasmodium, causes millions of deaths worldwide annually. Of the five Plasmodium species that can infect humans, Plasmodium falciparum causes the most serious parasitic infection. The emergence of drug resistance and the ineffectiveness of old therapeutic regimes against malaria mean there is an urgent need to better understand the basic biology of the malaria parasite. Previously, we have reported the presence of parasite-specific helicases identified through genome-wide analysis of the P. falciparum (3D7) strain. Helicases are involved in various biological pathways in addition to nucleic acid metabolism, making them an important target of study. Here, we report the detailed biochemical characterization of P. falciparum parasite-specific helicase 1 (PfPSH1) and the effect of phosphorylation on its biochemical activities. The C-terminal of PfPSH1 (PfPSH1C) containing all conserved domains was used for biochemical characterization. PfPSH1C exhibits DNA- or ribonucleic acid (RNA)-stimulated ATPase activity, and it can unwind DNA and RNA duplex substrates. It shows bipolar directionality because it can translocate in both (3'-5' and 5'-3') directions. PfPSH1 is mainly localized to the cytoplasm during early stages (including ring and trophozoite stages of intraerythrocytic development), but at late stages, it is partially located in the cytoplasm. The biochemical activities of PfPSH1 are upregulated after phosphorylation with PKC. The detailed biochemical characterization of PfPSH1 will help us understand its functional role in the parasite and pave the way for future studies.