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Introduction Chagas disease (CD), caused by the blood borne parasite Trypanosoma cruzi , affects 6–8 million people primarily in Latin America. Chronic CD leads to progressive damage in major organs, particularly the heart and gastrointestinal tract, with significant morbidity and mortality. Despite the gut serving as a key parasite reservoir, the immune response within this tissue during chronic infection remains poorly understood. This study investigates the role of double-negative (DN; CD3 + /CD4 - /CD8 - ) T cells in gut immunity during T. cruzi infection. Understanding their function could provide insight into mechanisms of parasite persistence and immune regulation in the gastrointestinal tract. Methods C57BL/6 mice were infected with a transgenic strain of T. cruzi Colombiana expressing a luminescence reporter. We collected tissues from infected mice during the acute (~30 days) and chronic phase (~90 days) of the disease and analyze them by bioluminescence imaging. We isolated cells from the gut lamina propria to identify the major immune cell types by multiparameter spectral flow cytometry. Colon tissue was also analyzed by fluorescence microscopy. Results We showed that DN T cells were the major lymphocyte population in this tissue by flow cytometry and fluorescence microscopy. The number of DN T cells increased significantly (~1.5 fold) during the acute phase and remained above control levels during the chronic infection. Two major subtypes were identified: (1) DN T cells with inflammatory phenotypes, which peaked in the acute phase and declined in the chronic phase but remained above control levels, and (2) DN T cells with regulatory phenotypes, which were elevated in both phases. Discussion Our findings highlight the critical role of colonic DN T cells in an experimental T. cruzi infection. Our results support a model in which DN T cells with inflammatory phenotypes may contribute to controlling the parasite burden in the gut during a chronic infection, whereas the sustained presence of DN T cells with regulatory phenotypes may help maintaining gut homeostasis but also create an anti-inflammatory niche that facilitates long-term T. cruzi persistence in this tissue. A better understanding the immune mechanisms occurring in reservoir tissues is important for developing improved therapies for Chagas disease.