AbstractThe eukaryotic parasite Plasmodium falciparum causes millions of malarial infections annually and drug resistance to common antimalarials confounds eradication efforts. Protein translation is an attractive therapeutic target that will benefit from a deeper mechanistic understanding. Translation initiation drives translational efficiency and is regulated by both cis and trans acting factors. P. falciparum mRNAs feature unusual 5’ untranslated regions suggesting that cis-acting sequences could play a significant role in determining translational efficiency. Here, we deployed in vitro translation to compare the role of cis-acting regulatory sequences in P. falciparum and humans. Using parasite mRNAs with high or low translational efficiency, the presence, position, and termination status of upstream start site, in addition to the base composition of the 5’ untranslated regions, were characterized. Within 130 nucleotide 5’ untranslated regions, upstream start sites were generally repressive but exhibited a nonadditive effect when combined, while the base composition of the two mRNAs demonstrated a more subtle role in regulating translational efficacy. Surprisingly, the effects of cis-acting sequences were remarkably conserved between P. falciparum and humans. While translational regulation is inherently complex, this work contributes toward a more comprehensive understanding of parasite and human translational regulation by examining the impact of discreet cis-acting features.