BackgroundIron is an essential element for cellular functions, such as energy metabolism.Trichomonas vaginalis, a human urogenital tract pathogen, is capable of surviving in the environment without sufficient iron supplementation. Pseudocysts (cyst-like structures) are an environmentally tolerated stage of this parasite while encountering undesired conditions, including iron deficiency. We previously demonstrated that iron deficiency induces more active glycolysis but a drastic downregulation of hydrogenosomal energy metabolic enzymes. Therefore, the metabolic direction of the end product of glycolysis is still controversial.MethodsIn the present work, we conducted an LC‒MS-based metabolomics analysis to obtain accurate insights into the enzymatic events ofT. vaginalisunder iron-depleted (ID) conditions.ResultsFirst, we showed the possible digestion of glycogen, cellulose polymerization, and accumulation of raffinose family oligosaccharides (RFOs). Second, a medium-chain fatty acid (MCFA), capric acid, was elevated, whereas most detected C18 fatty acids were reduced significantly. Third, amino acids were mostly reduced, especially alanine, glutamate, and serine. Thirty-three dipeptides showed significant accumulation in ID cells, which was probably associated with the decrease in amino acids. Our results indicated that glycogen was metabolized as the carbon source, and the structural component cellulose was synthesized at same time. The decrease in C18 fatty acids implied possible incorporation in the membranous compartment for pseudocyst formation. The decrease in amino acids accompanied by an increase in dipeptides implied incomplete proteolysis. These enzymatic reactions (alanine dehydrogenase, glutamate dehydrogenase, and threonine dehydratase) were likely involved in ammonia release.ConclusionThese findings highlighted the possible glycogen utilization, cellulose biosynthesis, and fatty acid incorporation in pseudocyst formation as well as NO precursor ammonia production induced by iron-depleted stress.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s13071-023-05842-w.