Serum-derived fatty acids are essential for the intraerythrocytic proliferation of Plasmodium falciparum in humans. We previously reported that only limited combinations of fatty acids can support long-term parasite culture, and palmitic acid (C16:0)/oleic acid (C18:1, n-9), palmitic acid (C16:0)/vaccenic acid (C18:1, n-7), or stearic acid (C18:0) are required in these combinations, implying that these fatty acids are key molecules for intraerythrocytic parasite growth (Mi-Ichi et al.2006). Here, we analysed profiles of parasitaemia changes as well as morphologies during the erythrocytic cycle and confirmed the importance of C16:0 and C18:1, n-9. We also provide evidence that C18:1, n-9 but not other C18 monoenoic or dienoic acids maintain the synchronicity of parasite development in serum-free medium when paired with C16:0, resulting in maintained exponential growth. Thus, C18:1, n-9 is indispensable for the intraerythrocytic proliferation of P. falciparum.