New effective antimalarial agents are urgently needed due to increasing drug resistance of Plasmodium falciparum. Phosphorothioate antisense oligodeoxynucleotides (ODNs) silencing of malarial topoisomerase II gene have shown to possess promising features as anti malarial agents. In order to improve stability and to increase intracellular penetration, ODNs were complexed with the biodegradable polymer chitosan to form solid nanoparticles with an initial diameter of ∼55 nm. The particle zetapotential depended on the chitosan/ODN mass ratio. Nanoparticles with mass ratio of 2:1 displayed a positive surface charge (+15 mV) whereas particles with 1:1 mass ratio were negatively charged (−20 mV). Additionally nanoparticles were found to protect ODNs from nuclease degradation. P. falciparum K1 strain was exposed to the chitosan/ODN-nanoparticles for 48 h in order to examine the effects of chitosan/antisense (AS) and chitosan/sense (S) oligodeoxynucleotide nanoparticles on malaria parasite growth. Both negatively and positively charged antisense nanoparticles as well as free antisense ODNs (in a final concentration of 0.5 μM) showed sequence specific inhibition compared with sense sequence controls. However, nanoparticles were much more sequence specific in their antisense effect than free ODNs. Nanoparticles with negative surface charge exhibited a significantly stronger inhibitory effect (∼87% inhibition) on the parasite growth in comparison to the positive ones (∼74% inhibition) or free ODNs (∼68% inhibition). This is the first study demonstrating the susceptibility of P. falciparum to antisense nanoparticles.