Niemann-Pick type C (NPC) disease is a lipid storage disorder characterized by accumulation of lipids in late endosome/lysosome (LE/LY) compartment. In our previous report we isolated membranes of the LE/LY compartment from NPC L1 skin fibroblasts with a mutation in the NPC1 gene and found that they were characterized by low fluidity which likely contributed to the impaired function of membrane proteins involved in storage and turnover of cholesterol. In this report we isolated lipid microdomains (DRMs) from membranes of various cellular compartments and observed an increased amount of DRMs in the LE/LY compartment of NPC L1 cells in comparison to control cells, with no change in DRM content in the plasma membrane. In addition, in the NPC cells, majority of the cholesterol-interacting protein, AnxA6, which participates in the transport and distribution of cholesterol, translocated to DRMs upon a rise in Ca2+ concentration. The mechanism of this translocation was further studied in vitro using Langmuir monolayers. We found that Ca2+ is the main factor which regulates the interaction of AnxA6 with monolayers composed of neutral lipids, such as DPPC and sphingomyelin, and may also determine AnxA6 localization in cholesterol and sphingomyelin enriched microdomains, thus contributing to the etiology of the NPC disease.