Flotillins are highly conserved integral membrane proteins which play a role in several key biological processes including vesicular trafficking, endocytosis, and cytoskeletal organization. Some studies have reported that flotillin-positive extracellular vesicles can be used as biomarker for AD diagnosis in blood samples. Due to its constitutive expression, flotillins can be expressed by many cell types. Our aim was to characterize the expression and localization of Flot1 in neurons and deduce the potential role of Flotillin-1 (Flot1) in AD. We use super-resolution microscopy methods such as Airy Scan and STED combined with western blotting to study Flot1 in primary hippocampal neurons from AppNL-F mice, a humanized knock-in mouse model of AD. In our initial immunolabelling studies, we found an increased expression and altered localization of Flot1 in AppNL-F neurons. With closer observation using STED, we found that Flot1 expression is vesicular in nature and colocalizes with APP in these vesicles. We found the number and size of the Flot1 vesicles to be altered both in soma and neurites in AppNL-F neurons. In conclusion, Flot1 showed an altered regulation in neurons derived from AppNL-F mice, which might be critical in endocytosis and vesicular trafficking of proteins related to AD pathology.