The circular genome of the Plasmodium falciparum apicoplast contains a complete minimal set of tRNAs, positioning the apicoplast as an ideal model for studying the fundamental factors required for protein translation. Modifications at tRNA wobble base positions, such as xm 5 s 2 U, are critical for accurate protein translation. These modifications are ubiquitously found in tRNAs decoding two-family box codons ending in A or G in prokaryotes and in eukaryotic organelles. Here, we investigated the xm 5 s 2 U biosynthetic pathway in the apicoplast organelle of P. falciparum . Through comparative genomics, we identified orthologs of enzymes involved in this process: SufS, MnmA, MnmE, and MnmG. While SufS and MnmA were previously shown to catalyze s 2 U modifications, we now show that MnmE and MnmG are apicoplastlocalized and contain features required for xm 5 s 2 U biosynthetic activity. Notably, we found that P. falciparum lacks orthologs of MnmC, MnmL, and MnmM, suggesting that the parasites contain a minimal xm 5 s 2 U biosynthetic pathway similar to that found in bacteria with reduced genomes. Deletion of either MnmE or MnmG resulted in apicoplast disruption and parasite death, mimicking the phenotype observed in Δ mnmA and Δ sufS parasites. Our data strongly support the presence and essentiality of xm 5 s 2 U modifications in apicoplast tRNAs. This study advances our understanding of the minimal requirements for protein translation in the apicoplast organelle.