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Pentatrichomonas hominis is an anaerobic flagellated protist that colonizes the large intestine of a number of mammals, including cats, dogs, nonhuman primates, and humans. The wide host range of this organism is alarming and suggests a rising zoonotic emergency. However, knowledge on in-depth biology of this protist is still limited. Similar to the human pathogen, Trichomonas vaginalis, P. hominis possesses hydrogenosomes instead of mitochondria. Studies in T. vaginalis indicated that hydrogenosome is essential for cell survival and associated with numerous pivotal biological functions, including drug resistance. To further decipher the biology of this important organelle, we undertook proteomic research in P. hominis hydrogenosomes. Lacking a decoded P. hominis genome, we utilized an RNA sequencing (RNA-seq) data set generated from P. hominis axenic culture as the reference for proteome analysis. Using this in-house reference data set and mass spectrometry (MS), we identified 442 putative hydrogenosomal proteins. Interestingly, the composition of the P. hominis hydrogenosomal proteins is very similar to that of T. vaginalis, but proteins such as Hmp36, Pam16, Pam18, and Isd11 are absent based on both MS and the RNA-seq. Our data underscore that P. hominis expresses different homologs of multiple gene families from T. vaginalis. To the best of our knowledge, we present here the first hydrogenosome proteome in a protist other than T. vaginalis that offers crucial new scholarship for global health, therapeutics, diagnostics, and veterinary medicine research. In addition, the research strategy used here using RNA sequencing and proteomics might inform future multi-omics research in other understudied organisms without decoded genomes.