ABSTRACTPlasmodium parasites and related pathogens contain an essential nonphotosynthetic plastid organelle, the apicoplast, derived from secondary endosymbiosis. Intriguingly, a highly conserved eukaryotic protein, autophagy-related protein 8 (ATG8), has an autophagy-independent function in the apicoplast. Little is known about the novel apicoplast function of ATG8 and its importance in blood-stage Plasmodium falciparum. Using a P. falciparum strain in which ATG8 expression was conditionally regulated, we showed that P. falciparum ATG8 (PfATG8) is essential for parasite replication. Significantly, growth inhibition caused by the loss of PfATG8 was reversed by addition of isopentenyl pyrophosphate (IPP), which was previously shown to rescue apicoplast defects in P. falciparum. Parasites deficient in PfATG8, but whose growth was rescued by IPP, had lost their apicoplast. We designed a suite of functional assays, including a new fluorescence in situ hybridization (FISH) method for detection of the low-copy-number apicoplast genome, to interrogate specific steps in apicoplast biogenesis and detect apicoplast defects which preceded the block in parasite replication. Though protein import and membrane expansion of the apicoplast were unaffected, the apicoplast was not inherited by daughter parasites. Our findings demonstrate that, though multiple autophagy-dependent and independent functions have been proposed for PfATG8, only its role in apicoplast biogenesis is essential in blood-stage parasites. We propose that PfATG8 is required for fission or segregation of the apicoplast during parasite replication.