Malaria's ability to rapidly adapt to new drugs has allowed it to remain one of the most devastating infectious diseases of humans. Understanding and tracking the genetic basis of these adaptations is critical to the success of treatment and intervention strategies. The novel antimalarial resistance locus PF10_0355 (Pfmspdbl2) was previously associated with parasite response to halofantrine, and functional validation confirmed that overexpression of this gene lowered parasite sensitivity to both halofantrine and the structurally related antimalarials mefloquine and lumefantrine, predominantly through copy number variation (CNV). Here we further characterize the role of Pfmspdbl2 in mediating antimalarial drug response in P. falciparum. Knockout of Pfmspdbl2 increased parasite sensitivity to halofantrine, mefloquine and lumefantrine, but not to unrelated antimalarials, further suggesting that this gene mediates parasite response to a specific class of antimalarial drugs. A single nucleotide polymorphism (SNP) encoding a C591S mutation within Pfmspdbl2 had the strongest association with halofantrine response and showed a high derived allele frequency among Senegalese parasites. Transgenic parasites expressing the ancestral Pfmspdbl2 allele were more sensitive to halofantrine and structurally related antimalarials than parasites expressing the derived allele, revealing an allele-specific effect on drug response in the absence of copy number effects. Finally, growth competition experiments showed that under drug pressure, parasites expressing the derived allele of Pfmspdbl2 out-competed parasites expressing the ancestral allele within a few generations. Together these experiments demonstrate that modulation of Pfmspdbl2 affects drug response in the malaria parasite.