Monoxenous trypanosomatid Strigomonas culicis harbors an endosymbiotic bacterium, which enables the protozoa to survive without heme supplementation. The impact of H₂O₂ resistance and symbiont elimination on intracellular heme and Fe²⁺ availability was analyzed through a comparison of WT strain with both WT H₂O₂-resistant (WTR) and aposymbiotic (Apo) protozoa. The relative quantification of the heme biosynthetic pathway through label-free parallel reaction monitoring targeted mass spectrometry revealed that H₂O₂ resistance does not influence the abundance of tryptic peptides. However, the Apo strain showed increased coproporphyrinogen III oxidase and ferrochelatase levels. A putative ferrous iron transporter, homologous to LIT1 and TcIT from Leishmania major and Trypanosoma cruzi, was identified for the first time. Label-free parallel reaction monitoring targeted mass spectrometry also showed that S. culicis Iron Transporter (ScIT) increased 1.6- and 16.4-fold in WTR and Apo strains compared to WT. Accordingly, antibody-mediated blockage of ScIT decreased by 28.0% and 40.0% intracellular Fe²⁺ concentration in both WTR and Apo strains, whereas no effect was detected in WT. In a heme-depleted medium, adding 10 μM hemin decreased ScIT transcript levels in Apo, whereas 10 μM PPIX, the substrate of ferrochelatase, increased intracellular Fe²⁺ concentration and ferric iron reduction. Overall, the data suggest mechanisms dependent on de novo heme synthesis (and its substrates) in the Apo strain to overcome reduced heme availability. Given the importance of heme and Fe²⁺ as cofactors in metabolic pathways, including oxidative phosphorylation and antioxidant systems, this study provides novel mechanistic insights associated with H₂O₂ resistance in S. culicis.