The glycosylphosphatidylinositol (GPI)-anchored mucins of Trypanosoma cruzi trypomastigotes play an important immunomodulatory role during the course of Chagas disease. Here, some biological activities of tGPI-mucins from four T. cruzi isolates, including benznidazole-susceptible (BZS-Y), benznidazole-resistant (BZR-Y), CL, and Colombiana, were evaluated. GPI-mucins were able to differentially trigger the production of interleukin-12 and nitric oxide in BALB/c macrophages and modulate LLC-MK2 cell invasion. The significance of these variations was assessed after analysis of the terminal α-galactosyl residues. Enzymatic treatment with α-galactosidase indicated a differential expression of O-linked α-galactosyl residues among the strains, with higher expression of this sugar in BZS-Y and BZR-Y T. cruzi populations followed by Colombiana and CL. Unweighted pair group method analysis of the carbohydrate anchor profile and biological parameters allowed the clustering of two groups. One group includes Y and CL strains (T. cruzi II and VI), and the other group is represented by Colombiana strain (T. cruzi I).