Sulfadoxine (SDX) and sulfamethoxazole (SMX) each inhibit the Plasmodium falciparum dihydropteroate synthetase (PfDHPS), and certain point mutations in this enzyme yield the drug-resistant parasite. Using a simple Escherichia coli model system, we describe here the effect of the recently reported novel K540N mutation in PfDHPS on the level of SDX/SMX resistance. The survival rate of the transformed E. coli (DHPS-deficient strain) under different SDX or SMX concentrations revealed that the K540N mutation confers a lower level of drug resistance than its contemporary K540E mutation. Further, SMX was more effective than SDX in the E. coli system.