Cystine-knot peptides are attractive templates in drug discovery due to a number of features they possess including their 3D conformation, physicochemical stability and synthetic tractability. Yet, their cellular uptake mechanisms remain largely unexplored. Recently, we demonstrated that the cystine-knot peptide EETI-II is internalized into cells and that its cellular uptake could be modulated by using a protein transfection reagent Xfect. However, the mechanism of Xfect-mediated cellular internalization of EETI-II remained unclear. Here, by using high resolution electron microscopy, we observe the formation of EETI-II-positive macropinosomes and clathrin-coated pits at early time points after treatment of cells with EETI-II/Xfect complexes. Internalized EETI-II subsequently accumulates in intracellular Xfect-induced detergent-resistant membrane compartments which appear to lack characteristic endosomal or lysosomal markers. Notably, Xfect enables the uptake of cell impermeable nuclear dyes into similar intracellular compartments that do not seem to deliver the cargo to the cytosol or nucleus. Altogether, our findings reveal mechanistic insights into the cellular uptake route of Xfect, and underscore the need for the development of effective tools to enhance the cytosolic delivery of cystine-knot peptides. Finally, our data illustrate that electron microscopy is a powerful approach for studying endocytic mechanisms of cell-penetrating peptides and their effects on cellular membranes.