Heme is an essential molecule for most organisms, yet some parasites, like Trypanosoma cruzi, the causative agent of Chagas disease, cannot synthesize it. These parasites must acquire heme from their hosts, making this process critical for their survival. In the midgut of the insect vector, T. cruzi epimastigotes are exposed to both hemoglobin (Hb) and free heme resulting from its degradation. Despite the importance of this nutrient, how different heme sources influence parasite gene expression remains poorly understood. Here, we showed that heme restitution either as hemin or Hb to heme-starved parasites induces an early and distinct transcriptional response in T. cruzi epimastigotes. Using RNA sequencing at 4- and 24-hours post-supplementation, we identified gene subsets commonly or uniquely regulated by each heme source, including genes putatively linked to heme acquisition and metabolism. We also presented here the first studies focused on CRAL/TRIO domain-containing protein (TcCRAL/TRIO), a novel heme responsive hemoprotein identified from this study. Our results provide a more detailed picture of T. cruzi biology and highlights heme acquisition as a promising point of vulnerability. These findings may ultimately contribute to the identification of potential molecular targets for the development of new therapeutic strategies against Chagas disease.