Background There are an estimated 260 million malaria cases and ∼600 000 deaths annually. Challenges to malaria eradication include the lack of highly effective and broadly applicable vaccines and parasite drug resistance. This is driving the need for new tools, including novel drugs and drug targets. The indoloquinolizidine alkaloid alstonine was previously shown to have in vitro activity against Plasmodium falciparum malaria parasites and a slow-action activity that is different from other slow-action antiplasmodial compounds such as clindamycin. Objectives To investigate the action of the antiplasmodial compound alstonine by validating a putative resistance mutation and determining whether the activity of alstonine is linked to the mitochondrial electron transport chain. Materials and methods In vitro evolution of resistance was used to generate alstonine-resistant P. falciparum, followed by whole-genome sequencing and CRISPR/Cas9 gene editing of wildtype parasites to validate a putative resistance-associated mutation. Links to mitochondrial function were assessed using oxygen consumption rate measurements and activity of alstonine in P. falciparum expressing the yeast dihydroorotate dehydrogenase. Results P. falciparum parasites were selected with ∼20-fold reduced sensitivity to alstonine compared to wild-type parasites. Whole-genome sequencing of alstonine-resistant P. falciparum sub-clones identified several mutations including a copy number variation and point mutation (A318P) in a gene encoding a putative inner-mitochondrial membrane protein (PfMPV17). Introduction of the A318P mutation into the PfMPV17 gene in wild-type P. falciparum yielded parasites with reduced alstonine sensitivity. While a direct link between alstonine action and mitochondrial respiratory function was not found, a transgenic P. falciparum line resistant to the cytochrome bc1 inhibitor atovaquone and pyrimidine synthesis inhibitor DSM265 had reduced sensitivity to alstonine. Conclusions These data demonstrate that PfMPV17 is linked to alstonine resistance and suggest that alstonine action is linked to the mitochondria and/or pyrimidine biosynthesis pathways.