Annexins are Ca²⁺-regulated phospholipid-binding proteins whose function is only partially understood. Annexin A4 is a member of this family that is believed to be involved in exocytosis and regulation of epithelial Cl⁻ secretion. In this work, fluorescent protein fusions of annexin A4 were used to investigate Ca²⁺-induced annexin A4 translocation and self-association on membrane surfaces in living cells. We designed a novel, genetically encoded, FRET sensor (CYNEX4) that allowed for easy quantification of translocation and self-association. Mobility of annexin A4 on membrane surfaces was investigated by FRAP. The experiments revealed the immobile nature of annexin A4 aggregates on membrane surfaces, which in turn strongly reduced the mobility of transmembrane and plasma membrane associated proteins. Our work provides mechanistic insight into how annexin A4 may regulate plasma membrane protein function.